Dynamic Bio-Nanoparticle Platforms

ABSTRACT

The invention in suitable embodiments is directed to dynamic bio-nanoparticle elements and bio-nanoparticle platforms employing such bio-nanoparticle elements. In one aspect, one or more elements of one or more types, formed from isolated, synthetic and or recombinant amino acid residues comprising in whole or in part one or more types of Clathrin and or Coatomer I/II proteins of one or more isoforms, execute one or more functions and or effect one or more ends, in vivo and or in vitro.

FIELD OF THE INVENTION

The invention relates generally to the field of nanoparticles, and morespecifically, in one embodiment, to dynamic bio-nanoparticle elementsformed from materials comprised of self-assembling protein molecules,which are capable of executing one or more functions and or effect oneor more ends, in vivo and or in vitro. In another invention embodiment,the invention relates to a multifunction nanoscale bio-nanoparticleplatform, such as a biomedical platform, bio-molecular platform,electronics platform, information processing platform, and the like,using such dynamic bio-nanoparticle elements.

BACKGROUND OF THE INVENTION

Structures at the nanoscale are sometimes referred to as nanoparticles.Some nanoparticles comprise cage elements that form cavities and orcomprise vesicle elements; examples of which in the prior art teachelements such as nano-carbon endohedral cages (Fullerenes); capsids, theprotein shell of a virus; liposomes; lipids; heat shock proteins;ferritins; vault ribonucleoprotein particles; Clathrin protein cages;and Coatomer I/II protein cages, among other various cage- orvesicle-forming elements. Additionally, prior art teaches that proteincage elements can coat vesicle elements; for example, Clathrin andCoatomer coated vesicles (CCV's). Additionally, prior art teaches thatone or more types of cargo elements can be located internally withrespect to a cage and vesicle element.

A cavity forming protein cage and a cage coated vesicle implementationis taught in issued U.S. Pat. No. 7,393,924 (Jul. 1, 2008, Vitaliano etal.) The cage and cage coated vesicle elements are formed in vitro froma plurality of isolated Clathrin/Coatomer protein subunits. As taught inU.S. Pat. No. 7,393,924, the enhanced functionalization capabilities ofthe isolated Clathrin and Coatomer I/II protein molecules enable anumber of properties and features that make them superior to other cageand cage coated vesicle elements in the prior art.

But the instant invention teaches nanoscale element fabrication,assembly, operation, behavior and properties that are unique from priorprotein art that encompasses various types of cavity-forming cagestructures formed in vitro from a plurality of self-assembling subunits.For example, a fully formed Clathrin cage element as taught in U.S. Pat.No. 7,393,924, and generally speaking taught in other Clathrin art, iscomprised of a plurality of 3-legged triskelia, each triskelion having 6protein subunits; 3 Clathrin heavy chain and 3 Clathrin light chainsubunits.

In marked contrast, the instant invention teaches that complete cagescomprised of a plurality of 3-legged triskelia are not required tocomprise one or more types of efficacious elements. Instead, in its mostessential embodiment the instant invention teaches one or more nanoscaleelements of one or more types formed from isolated, synthetic and orrecombinant amino acid residues comprising in whole or in part one ormore types of Clathrin and or Coatomer I/II proteins of one or moreisoforms, including cloned isoforms. These isoforms with their differingamino acid sequences comprise (in this example, humans) the varioustypes of Clathrin heavy chains, the various types of Clathrin lightchains, encompass the distinct heavy chain and light chain segments anddomains, and in the case of Coatomer, comprise and encompass its domainsand subunits, with different combinations of the latter known to existwithin Coatomer complexes. Examples of amino acid sequences comprisingClathrin and Coatomer proteins, and their respective isoforms are listedin Tables 1-11. Accordingly, one or more instant invention embodimentsmay also comprise minimalist, non-cage elements of one or more types.The minimalist element structure afforded by the instant inventionaffords a much broader and richer variety of element configurations andembodiments than those taught in prior Clathrin or other protein cageart.

For example, freed of the constraints of only forming cavity-formingprotein cages in vitro, one or more non-cage invention elements may alsoform one or more other types of nanoscale elements and structures,enabling new classes and types of applications. Example non-cageembodiments include, but are not limited to, functionalized nano-tubulestructures; protein-based nano-dendrimers suitable for biomedical andbio-molecular applications; and self-assembling, stable, bioactive,protein-based, hydrogel nanoparticles (nanogels). In other embodiments,one or more nanoscale elements and structures may be additionally formedand comprised of one or more non-invention elements of one or moretypes. Such structural plasticity and flexible element functionality arenot taught in prior protein cage art.

Prior art often teaches one or more types of protein cages that carryone or more types of additional elements, e.g., cargo, to enable overallfunctionality and produce efficacious results. However, unlike priorart, the instant invention teaches, in one embodiment, one or morenon-cage or cage elements may carry no additional elements like cargo,yet still can comprise inherently efficacious elements of one or moretypes, like drug elements, but not limited to. In one embodiment, one ormore invention elements operating alone and without any additionalelements such as cargo and the like comprise unique new types ofinherently efficacious agents and elements that are distinctly differentin behavior and functionality from prior art, and their unique featurescorrespondingly enable new types of applications.

In another embodiment, one or more elements and or their additionalelements in whole or in part may require only minimal functionalizationto be efficacious; e.g., they may not require PEGylation or other typesof functionalization to operate effectively.

In another embodiment, one or more elements carry one or more types ofcargo and the cargo acts as the efficacious element. In anotherembodiment, one or more elements together with cargo elements act inefficacious concert.

In another embodiment, one or more elements are penetrating elementsthat enter one or more cells and gain access to the cytosol andintracellular elements of one or more types, including one or more cellorganelles. Such elements may, in one embodiment, require minimalfunctionalization. In another embodiment, one or more elements maycomprise one or more membrane fusion elements. These various featuresare not taught in prior protein cage art. In one embodiment, using cellcrossing techniques yield efficacious cancer treatments, gene therapy,and the like.

Further, in cage, cavity, and vesicle prior art, one or more types ofadditional elements, e.g., cargo, are often inserted into a complex,fully formed structure, a sometimes difficult and laborious process. Butthe invention, in one or more embodiments, teaches that using utilizingnon-cage elements of one or more types makes the addition of one or moreelements less difficult as there is no insertion process into a cage,cavity, or vesicle to contend with. In another embodiment, additionalelement functionalization is simplified by decorating just the externalsurface of a cage, a feature not taught in prior Clathrin art.

In another embodiment, one or more assay, diagnostic, therapeutic, andprosthetic applications and the like can be performed ensemble using thesame bioengineered element.

These various functionalization capabilities enable a highly flexiblenano-platform that features improved stability, rigidity, functionalityand loading capacity relative to other nanoparticles, and beingcomprised of ubiquitous proteins, features low antigenicity in one ormore embodiments. In one illustrative embodiment, one or more elementsmay be harmlessly dissolved, passed, and or excreted from the body.

In one embodiment, the current application teaches one or more elementscomprising one or more types of hybrid elements and arrangements, whichcan produce efficacious results. In one embodiment, one or moreinvention elements are conjugated to natural biological/molecularelements, like cells, but not limited to, forming one or more types ofhybrid elements in vitro and or in vivo. Such hybrid elements mayoperate alone or with additional elements, e.g., with cargo. In anotherembodiment, such hybrid elements may fuse in vitro and or in vivo withnon-invention elements, such as those comprising natural elements incells, but not limited to. This type of hybrid/fusion capability andflexibility is not taught in the prior art.

In another embodiment, the current application teaches one or moreelements, functioning alone or with one or more additional elements,which comprise efficacious replacements for one or more elements of oneor more types, including non-invention elements. In one embodiment, oneor more elements may replace one or more types of naturally occurringcell elements, to efficacious effect. This replacement capability is nottaught in the prior art.

In one embodiment, the instant invention teaches one or more elements,functioning alone or with one or more additional elements, whichcomprise one or more cellular repair elements, of one or more types; acapability not taught in the prior art. In another embodiment theelements are cellular regeneration elements.

Prior art also does not teach that cage, vesicle elements, or theirvarious subunit elements efficaciously operate in the extra-cellularspaces, e.g., in the synaptic spaces between neurons. But the instantinvention teaches one or more types of elements capable of suchextracellular operation, including for the in situ remediation, removaland or sequestration of undesirable organic and or non-organic elements.

The invention further teaches a biological model that is consistent, notfrom the complete cage element level up, but from the minimalist,non-cage element level up, in vitro and in vivo, making drug discoverysafer, more efficacious, more time and cost effective, and overall, amuch more rapid process than prior art.

In another embodiment, one or more elements may comprise one or moretypes of minimalist, non-cage elements than that taught in prior art fordoing clinical trials of one or more types of agents, including theirtargeted agent delivery, including high precision dosing.

In one embodiment, the instant invention teaches one or more elementsthat in whole or in part execute one or more types of actions forcreating, spawning, comprising, modifying, repairing, regenerating,reassembling, and or control and regulation of one or more cells,cellular elements, cell organelles, including like actions and behaviorsinvolving cellular processes such as endocytosis, exocytosis, mitosis,trafficking and signaling, communication between cells, receptorupregulation and downregulation, other behaviors, and the like. Failuresand defects in any of these cellular elements and processes can lead todiseases, for example, cancer. This type of efficacious behavior is nottaught in prior art, including in protein cage art.

In one invention embodiment, one or more elements, with or withoutadditional elements, and in some embodiments with minimalfunctionalization, enter the central nervous system, including passingthe blood brain barrier (BBB) for efficacious effect. Although differentprotein cage types, e.g., viruses, have been investigated as MRInano-probes, some types of these cages in prior art did not cross theBBB, and other types in prior art were shown to be immunogenic aftercrossing the BBB.

In one embodiment, the invention enables post administration delivery ofone or more types of agents into the CNS in 30 minutes or less. In otherembodiments, delivery of agents occurs in 30 minutes or more. In anotherembodiment, agents operate in the inter-neuronal spaces. Prior art doesnot teach such flexible CNS delivery arrangements.

The instant invention teaches self-directing, self-replicating,self-adapting, self-repairing, self-regulating, and or self-regeneratingmethods for one or more minimalist, non-cage elements, which can alsoperform on-the-fly target prioritization. Prior protein cage art doesnot teach such self-modifying methods at a minimalist, non-cage elementlevel.

Prior art does not teach enabling and or utilizing quantum mechanicaleffects using just one or more minimalist, non-cage elements. But in oneembodiment, the instant invention teaches enabling and utilizing suchquantum mechanical effects.

The instant invention also teaches a plurality of elements of one ormore types that can, in one illustrative embodiment, function asbiomedical platform and the like, and in another example embodiment,function as a biomolecular component platform and the like, or as aninformation processing platform that can carry out algorithmicallydefined actions, and other types of platforms.

Thus, there exists a need for an improved bio-nano-structure elementthat overcomes the limitations in the prior art for various types of invivo and in vitro applications.

SUMMARY OF THE INVENTION

The invention, in one aspect, remedies the deficiencies of the prior artby teaching modifiable, interactive, dynamic bio-nanoparticle elements,some of which may comprise minimalist, non-cage embodiments, with orwithout one or more additional elements of one or more types located onand or in one or more elements; whose applications, in one or moreembodiments, focus on forming in whole or in part one or more nanoscaleelements and structures of one or more types that execute one or morefunctions and or effect one or more ends in vivo and or in vitro.

In one illustrative embodiment, the invention is an improvement overother in vivo biodegradable polymer nanospheres, liposomes, lipids,capsids agent delivery systems, as well as endohedral Fullerenes andother bio-nanoparticles in the prior art because the invention enables,among other unique features:

-   -   Simplified nanoscale fabrication    -   Simplified cargo and other element type attachment.    -   Cell and organelle crossing, and or membrane fusion.    -   Low antigenic, “green” nanotechnology.    -   Interaction, control, and regulation of cellular processes, like        endocytosis, exocytosis, mitosis, trafficking and signaling,        communication between cells, receptor upregulation and        downregulation, other cellular behaviors, and the like.    -   Entering the CNS, including passing the blood brain barrier, and        in some cases, in less than 30 minutes post administration.    -   One or more elements that carry no additional elements, like        cargo, and operating alone produce an efficacious effect, acting        like a drug, for example.    -   Hybrid invention elements comprised of one or more types of        non-invention elements, e.g., natural cell elements.    -   Self-modifying, orchestrated actions at a minimalist, non-cage        level using natural control laws that govern biological        elements.    -   Methods and behaviors defined by algorithms.

In one particular embodiment, one or more of self-assembling Clathrinand or Coatomer elements are functionalized, modified and orbioengineered using commercially available biotechnology tools and othertools and techniques known in the art, which makes the invention moreversatile and cost-effective than the existing art.

In another embodiment, one or more elements are also comprised of one ormore non-invention elements, e.g., one or more invention elements areconjugated to natural biological/molecular elements, like cells, but notlimited to, forming one or more types of hybrid elements in vitro and orin vivo.

In one illustrative embodiment, one or more elements can be of anysuitable size. According to an illustrative embodiment, one or moreelements are nanoscale elements.

The invention, in one embodiment, teaches one or more elements thatdynamically and interactively respond to changing in vivo and or invitro environments; e.g., change of pH, temperature, biochemical, orbiological conditions, and the like.

In one embodiment, one or more elements, in one or more configurations,utilize self-directing, self-adapting, self-assembling, self-repairing,self-regenerating, self-regulating, and or self-replicating methods.

In one embodiment, one or more elements, in one or more configurations,utilize goal directed methods.

In one embodiment, one or more elements utilize, respond to, and orexhibit one or more effects, such as quantum mechanical, mechanical,photonic, acoustic, electrical, biochemical and chemical, and the like.

The invention, in one embodiment, provides one or more elements thatmaintain structural and or functional integrity long enough to do usefulwork, in vivo and or in vitro.

According to one feature, one or more elements re-supply, repair,reassemble and or regenerate defective, destroyed and or inoperableelements of one or more types, including non-invention elements, in vivoand or in vitro.

In another embodiment, one or more types of elements, unlike othernanoparticles in the art; such as nano-carbon, virus capsids, as well asnano-coating elements like polysorbate; may exhibit no or limitedimmunogenic, toxic, and or environmental impact effects, and dependingon cargo and other element type also may require little or nofunctionalization,

In another embodiment, elements maintain structural integrity at roomtemperature in vitro and vivo, which eliminates the need for elaboratestructure stabilizing mechanisms, like cooling systems.

Another advantage of the invention is that its protein material does notexhibit extreme hydrophobicity.

According to another feature, one or more elements are protected fromthe external environment, and the invention is stable with respect todissociation and any element toxicity is sequestered from thesurrounding in vivo and or in vitro environment.

In some embodiments, bonding and or attachment methods of one or moretypes, e.g., covalent, non-covalent, and any other bond type that can beexplained by quantum theory, are used to directly attach one or moreelements, internally or externally to one or more other elements in anordered arrangement.

In one embodiment, one or more elements each may bond with one or moreother elements, of one or more types, including invention andnon-invention elements.

In one embodiment, one or more elements may additionally have located onand or in them one or more cargo elements of one or more types, formedfrom one or more types of molecules.

In another embodiment, the invention features precise, highly orderedplacement of additional elements, like cargo elements, with minimalinter-element spacings on one or more elements and structures.

In one embodiment, one or more cargo elements comprise natural,isolated, synthetic and or recombinant elements.

In one embodiment, one or more cargo carrying elements include in wholeor in part one or more non-invention elements of one or more types.

In one embodiment, one or more cargo elements and or cargo carryingelements comprise hybrid elements of one or more types.

In one embodiment, one or more elements of one or more types do notcarry cargo elements.

In one embodiment, nanoscale ensembles comprising one or more types ofelements allow for a large variety and number of possible cargo elementconfigurations.

In one embodiment, one or more elements may additionally have located onand or in them one or more elements such as ligand elements, receptorelements, adaptor protein elements, and the like, formed from one ormore types of molecules, which may also comprise one or more hybridelements formed from one or more non-invention elements.

In another embodiment, one or more elements may be comprised of one ormore elements derived in part from one or more types of elements, forexample, but not limited to, an amino acid sequence derived from aClathrin or Coatomer protein.

In another illustrative embodiment, one or more elements, in one or moreconfigurations, are coated in whole or in part with chemicals, metals,biomaterials, and or other substances, of one or more types.

In another illustrative embodiment, one or more elements, in one or moreconfigurations, comprise one or more organic, inorganic, and orsynthetic material elements, of one or more types, in one or more formsand or phases, in whole or in part

In one embodiment, one or more elements are radiation shielded, radiofrequency (RF) shielded, thermally shielded, chemically shielded, andthe like, in whole or in part, and in one or more configurations.

In various embodiments, one or more elements may be of more than onefunctionalization type, and or express more than one type offunctionality.

In one embodiment, one or more elements in whole or in part may requireminimal or no functionalization to be efficacious elements, like a drugand the like, but not limited to.

In another embodiment, one or more elements in whole or in part compriseone or more structures, of one or more types.

In another embodiment, one or more elements in whole or in part comprisea shape programmable and or shaped scaffolding system via which one ormore elements of one or more types form one or more structures with oneor more types of shapes and or functions.

In one embodiment, one or more elements act as one or more types ofefficacious replacements for one or more other elements, includingnon-invention elements, in vitro and or in vivo, e.g., act asreplacements for one or more natural elements commonly found in cells,but not limited to. This type of replacement functionality is not taughtin prior art, including protein cage art.

According to one approach, various self-assembling and self-directedmethods are employed. Elements and or their platforms can be formed fromthe bottom-up, one element at a time. Another advantage of bottom-upfabrication is that it reduces the amount of superfluous material thatsurrounds each cargo element, reducing the element's exposure tocontaminant background radiation and thereby improving the functionaleffectiveness of the element.

In one embodiment, the instant application teaches one or more nanoscaleelements of one or more types formed from isolated, synthetic and orrecombinant amino acid residues comprising in whole or in part one ormore types of Clathrin and or Coatomer I/II proteins of one or moreisoforms, including cloned isoforms. The efficacious elements maycomprise minimalist, non-cage forming elements in one or moreembodiments. In other embodiments, one or more Clathrin or Coatomer cageelements comprise efficacious elements.

In one embodiment, one or more elements may additionally comprise ahybrid molecular element formed from one or more other types ofmolecules.

The instant invention teaches that in one or more non-cage elementembodiments it features unique types of dynamic properties andcapabilities not found in fully self-assembled, cavity-forming cagestructures as taught in the prior art.

In one embodiment, an element is comprised of one or more 3-leggedtriskelia, each triskelion having 6 protein subunits; 3 Clathrin heavyand 3 light chain subunits. In another example embodiment, the instantinvention teaches one or more configurations as being comprised of only3 Clathrin heavy subunits or only 3 light chain subunits. In anotherillustrative embodiment, configurations comprised of less than 3Clathrin heavy or 3 light chain subunits are enabled. In anotherembodiment, the invention teaches elements comprising in part one ormore types of Clathrin and or Coatomer I/II proteins of one or moreisoforms

Likewise, the invention teaches one or more highly flexible elementembodiments formed from Coatomer I/II proteins. In one embodiment, oneor more nanoscale elements of one or more types are formed fromisolated, synthetic and or recombinant amino acid residues comprising inwhole or in part one or more types of Coatomer I/II proteins of one ormore isoforms, including cloned isoforms. Components of both COP1 andClathrin-adaptor coats share the same structure and the same motif-basedcargo recognition and accessory factor recruitment mechanisms, whichleads to insights on conserved aspects of coat recruitment,polymerization and membrane in deformation. These themes point to theway in which evolutionarily conserved features underpin these diversecell pathways.

In one example embodiment, one or more elements comprised of Coatomer(COPI and COPII) proteins, which can efficaciously act alone or withadditional elements, are used instead of Clathrin proteins, preferablyin those applications where Coatomer characteristics would be moredesirable than those of Clathrin. Coatomer I/II protein elements may, inone or more embodiments, be comprised of one or more alpha, beta, beta′,gamma, delta, epsilon and or zeta subunits. Different combinations ofthese subunits are known to exist within Coatomer complexes. Accordingto an illustrative embodiment, a Coatomer subunit is a nanoscaleelement. In one invention embodiment, Clathrin and Coatomer elements andone or more methods may be used together in one or more configurations,taking advantage of their respective capabilities.

Freed from the constraints of only assembling into cavity forming cagesin vitro, one or more non-cage elements of one or more types mayself-assemble into one or more other types of complex elements and ormaterial forms, enabling new classes of applications. For example, butnot limited to, using techniques known in the art, bioengineered strandsof Clathrin and or Coatomer proteins form functionalized nano-tubules(Zhang, et al. 2007) for biomedical applications and bio-molecularcomponents. In another bioengineered embodiment, invention elementscomprise repeatedly branched, highly symmetrical structures, formingprotein-based nano-dendrimers suitable for biomedical and bio-molecularapplications. In another embodiment, self-assembling, stable, bioactive,protein-based, hydrogel nanoparticles (i.e., nanogels), some withtunable structural properties, are enabled. Generally, hydrogels are ofinterest to the biomedical field, e.g., for treating trauma, because thehydrated networks can provide a physiological environment wherebiological species can survive or grow. In other embodiments, one ormore other types of non-cage forming structures, elements, and forms ofmaterials comprised of invention elements are formed using techniquesknown in the art.

Unlike cage, cavity, and vesicle systems in the prior art where one ormore additional elements, e.g., cargo, are inserted into a complex,fully formed structure; a sometimes difficult and laborious process; theinvention, in one embodiment, teaches that it can be functionalized withone or more additional elements at a much more fundamental nano-elementlevel, e.g., by using non-cage elements of one or more types formed fromamino acid residues of Clathrin or Coatomer proteins. Suchfunctionalized, minimalist elements may further self-assemble in vitrointo one or more nanoscale structure elements, including cages. Thismakes the addition of one or more elements easier and simpler as thereis no insertion process into a completely formed cage, cavity, orvesicle. In another embodiment, additional element functionalization issimplified by decorating just the external surface of a cage.

According to one illustrative configuration, one or more types ofelements, such as cargo elements, may interfere with the invention'soverall operation if carried in the same element as other element types.Instead, the problematic elements are carried in a separate element thatexclusively carries non-interfering elements, thereby inhibitingdisruptive interference of invention operations. Such non-interferingelements may be functionally and or physically linked with otherelements carrying other element types.

In one embodiment, one or more elements efficaciously operate alone andcarry no additional elements, e.g., cargo. In one embodiment, such soloelement functionality produces a unique new type of efficacious element,and its unique features correspondingly enable new types ofapplications.

Some embodiments include a molecule having an unpaired electron, atransition metal ion, which can be found in the active centers of manyproteins (metalloproteins), or a material having any defect thatproduces an unpaired electron.

According to one in vivo application for enhanced medical imaging,paramagnetic lanthanide, transition metal ion complexes, and the likeare cargo elements that modify the NMR relaxation times of nearby protonnuclei of H₂O molecules, leading to brighter images and enhancedcontrast between areas comprising the contrast agent and the surroundingtissues.

In another illustrative embodiment, one or more elements accept freeradical molecules such as nitroxide molecule spin labels for electronparamagnetic resonance (EPR) based invention applications.

In another illustrative embodiment, one or more elements accept and orcomprise one or more types of labels and assay strategies, andinstruments for detection of one or more such labeled and or assayelements may include, but are not limited to: fluorescence and confocalmicroscopy, flow cytometry, laser scanning cytometry, fluorescencemicroplate analysis and biochips, immunoassay systems, nucleicacid-based diagnostics, and the like. In various embodiments, one ormore elements meet and or surpass the requirements for label and assaysensitivity, accuracy and convenience.

In another embodiment, one or more types of elements such as comprisingin whole or in part one or more large molecule elements, small moleculeelements, cargo elements, agent elements, device elements, drugelements, and the like, enter the CNS, including passing the blood brainbarrier, in 30 minutes or less and or in 30 minutes or more, postadministration, and, depending on cargo and other element type, mayrequire minimal functionalization for such element passage.

In some configurations, one or more elements comprise a cargo element,while in other configurations they comprise multiple elements, of one ormore types. In some configurations, one or more or each of the elementsand or cargo elements is a metal, and or may include one or more metals.Alternatively, each of the elements and or cargo elements is or includesnon-metal elements. In other embodiments, elements and or cargo elementsare exclusively non-metal elements that may include gases, as well asother elements like biological elements, drugs, optics, polymers, etc.In another embodiment, one or more elements and or additional elementscomprise one or more types of material forms, including a solid, gas,vapor, crystal, and the like. In another embodiment one or moreinvention and or non-invention elements, in one or more combinations,comprise one or more types of isolated, synthetic and or recombinantelements.

An invention element, in one functionalized configuration, includesreceptor molecules; natural, isolated, synthetic and or recombinant, forcapturing and ordering the placement of one or more elements, like cargoelements, on one or more elements.

An invention element, in another functionalized configuration, includesadapter molecules; natural, isolated, synthetic and or recombinant,disposed between the receptor molecules and one or more elements tocouple the receptor molecules to another element, like to a cargoelement.

An invention element, in one functionalized configuration featuresligands, natural, isolated, synthetic and or recombinant, includingdrugs, of one more types attached to receptors and or adapter proteinelements.

In one configuration, one or more elements, of one or more types, areattached to one or more types of amino acids on one or more elements.

In another configuration, biotin-avidin is used as a coupler of one ormore elements, of one or more types, to one or more elements of one ormore types.

In another configurations, PEGylation, a cross-linker, molecular bridge,molecular tether, and the like are used to attach one or more elements,of one or more types, to one or more elements of one or more types.

In one example, molecules of one or more types are attached to a shortmolecular tether to one or more elements via site directed substitutionmutagenesis, followed by reaction of a unique amino acid group with aspecific molecular label.

In another embodiment, free radicals, toxic elements, other types ofundesirable elements and the like circulating within an in vivoenvironment are scavenged via molecular tethers, via other elements ofone or more types attached to one or more invention elements, and or viadirect binding to one or more elements.

In another embodiment, the invention takes full advantage of proteinflexibility and plasticity to create elements of one or more types thatare bonded, fastened, fused, and or affixed to one or more otherelements, of one or more types.

In one illustrative embodiment, one or more elements and or bondedelements are coated in whole or in part with other elements, such aschemical, biological and or metallic materials, and the like. Thecoating elements may be or include organic, inorganic, and or syntheticmaterials, or a combination thereof.

In another invention embodiment, site directed mutagenesis is used toincorporate one or more elements, of one or more types, into one or moreother elements, of one or more types.

In one embodiment site-directed mutagenesis using one or more types ofprimer; including its reverse complement; are used to insert one or moreDNA sequences of one or more types into one or more coding regions ofone or more elements.

In another embodiment, cloning is done of one or more genes encoding oneor more elements. In another embodiment, one or more amino acids and ortheir encoder gene are controlled, regulated, modified, and the like, byone or more methods known in the art to produce an efficacious effect,in vivo and or in vitro.

In one embodiment, one or more elements of one or more types comprisetargeted and or non-targeted drug elements, biological elements, otherforms of healthcare elements, including cosmetic elements, in one ormore configurations or combinations, for diagnosing, remedying,inhibiting, mitigating, curing, and or preventing one or more types ofdiseases, infections, physical or mental trauma, other forms of physicaland mental afflictions, and the like, of one or more types, includingtypes featuring minimal immunogenic and or toxic effects.

In one embodiment, one or more elements are used as a means forevaluating drug advancement and efficacy.

The invention teaches a biological model and or method that isconsistent from a minimalist component level up, e.g., amino acidresidues comprising in part one or more Clathrin and or Coatomer I/IIproteins of one or more isoforms, making drug discovery safer, moreefficacious, more time and cost effective, and overall, a much morerapid process.

In one personalized medicine embodiment, the invention reduces drug sideeffect profiles and or produces greater agent efficacy, as well asexcludes agents that may have no efficacy in a particular individual.The invention, in one embodiment, provides for individual patientfactors such as genotype, phenotype, age, gender, ethnicity etc., to betaken into account by one or more elements and factored into dosing andadministration consideration.

In one embodiment, one or more elements comprise one or more types ofpluripotent stem cells and or comprise one or more stem cell deliverymethods.

According to one feature, one or more elements may be or include one ormore research, therapeutic, diagnostic, vaccine, assay, and orprosthetic agents, in one or more configurations, and thereby constituteone or more types of biomedical elements. Such biomedical elements maybe, for example, nano-structured and/or include chemical, biologicaland/or metallic materials. The biomedical elements may be or includeorganic, inorganic, and or synthetic materials, or a combinationthereof.

Medical, biomedical, bioengineered, and or biological applications andplatforms of the instant invention may include, but are not limited to,imaging; sensor; genetic and protein assay; diagnostic; drugs and drugdelivery; prosthetic; inter- and extra-cellular tissue; whole organ;circulatory system; medical device; implantable defibrillator;pacemaker; coronary stents; angioplasty device; and other likeapplications.

In one embodiment, one or more elements comprise one or moreapplications that perform analysis, of one or more types, of disordersof complex inheritance.

In one embodiment, one or more elements comprise one or moreapplications that perform analysis, of one or more types, ofpharmacologic therapy.

In one embodiment, one or more elements comprise one or more types ofprognosis and therapy selection—“theradiagnostics”.

In one embodiment, one or more elements comprise one or more genomicapplications of one or more types.

In one embodiment, one or more elements comprise one or more oncologyapplications of one or more types.

In one or more embodiments, one or more elements may use routes ofadministration comprising one or methods of one or more types, such asthose defined by CDER Data Element Number C-DRG-00301 in the US FDA DataStandards manual. Routes of in vitro administration of one or moreelements may also comprise one or more forms.

In one or more embodiments, one or more pharmaceutical and drugformulations of one or more types are used, in whole or in part, such astablet, capsule, soft galantine capsule, topical, injections, eye drops,syrups and liquids, soap and cosmetics, birth control device, and thelike, but not limited to, as well as one or more types of biologics,chemical compounds, water soluble compositions, and the like, but notlimited to. In vitro formulations may also comprise one or moreformulations of one or more types in one or more embodiments.

According to one feature, one or more elements respond to one or moreexternal and/or internal stimuli, which can be, for example, mechanical,chemical, biochemical, biological, metabolic, covalent, non-covalent,photonic, sonic, acoustical, thermal, fluidic, electromagnetic,magnetic, radioactive, quantum mechanical, or electrical in nature.Examples of such a stimulus response is altering a cargo element carriedby an element; the altering of the element itself; causing changes incellular process like endocytosis, exocytosis, mitosis, trafficking andsignaling, and the like, including other conformational changes.

In another embodiment, photonic energy impacting one or more elementsproduces electrical current, and or photonic energy, e.g., a laser.

In general, in another embodiment, one or more element and or platformare physically and/or functionally cooperative with other suitable typesor forms of elements, agents, organisms, materials, substances,components, devices, and or systems, including non-invention elements,in vitro and/or in vivo.

The invention, in one embodiment, provides for a plurality of elementscomprising aggregated, complex self-assembled nanoscale structures thatdynamically bind together one or more types of endogenous, exogenous,homogeneous, and or heterogeneous elements into one or more complexelements, which also may have one or more payload types.

The invention, in one embodiment, provides a capability for in vivo andin vitro integration of one or more types of elements into otherelements, devices and mechanisms, some of which may also benon-invention elements, that also may be linked together functionally orlogically, including with other devices and or operators, locally or ata distance, significantly enhancing the overall capabilities of theinvention.

In one embodiment, the invention provides for the ability of one or moreelements to track, recognize, attack and or destroy multiple targets onthe fly, in vivo and in vitro, using dynamic target prioritization for asingle element type and or multiple element types.

In one application, one or more elements, including cargo elements,comprise one or more types of targeted agent delivery systems and oragents in vivo or in vitro, including high precision dosing, using, asappropriate, ligands, targeting moieties, and or other vectors. In oneapplication, one or more targeted elements comprise one or moreresearch, remedial, inhibitory, mitigation, preventive, prosthetic,assay, and or other type of bio-molecular agent or device, in one ormore combinations, and may altogether comprise a unified element and orplatform.

The invention, in one embodiment, provides for a method for targeteddelivery systems that leverage and utilize biological control laws andthat may act as self-directed systems.

According to another invention embodiment, one or more targeted elementsmay use molecular-imprint technology, which is used for the productionof molecule-specific cavities that mimic the behavior of receptorbinding sites, without the temperature sensitivity of natural systems.

According to another feature, biodegradable films may also be used as apliable template for one or more targeted elements, which are pressedinto a biodegradable film and then removed, leaving a physical mold ofthe element's shape. The film can then be hardened and used by anelement to detect a particular element, which may be, but is not limitedto, a particular receptor, protein, or cell, since its complex imprintshape on the film will bind only to that particular biological element.

In one embodiment, the invention provides for a targeting system usingbiodegradable nanocapsules for delivery of one or more elements in vivoor in vitro.

In another application, a nanoscale platform comprised of a plurality ofelements performs molecular-level and or cellular-level target siteloitering, monitoring, repair, construction and or dynamic, interactivecontrol and regulation of biological systems, in vitro and in vivo.

In another embodiment, one or more elements, including in whole or inpart one or more non-invention elements, operating alone or with one ormore additional elements, comprise one or more types of membrane fusionelements. In one embodiment, the resulting biological processes andinteractions from such fusion may lead to a series of controlled,regulated, extended, modulated, purposefully, and or self-directedmethods and or behaviors of elements.

In one example embodiment, one or more elements in whole or in partexecute one or more types of actions involving conformational changes,bonding, attachment, and or the fusion of one or more elements to a cellmembrane, one or more of which actions may lead to changes in cellularprocesses, such as endocytosis, exocytosis mitosis, trafficking andsignaling, and the like, and or enable the precise dispatch andsequenced delivery of selected agents from an element to a target cell.Alternatively, a series of interlocking steps between a part of a cellmembrane, and all, or a subset of the materials comprising an elementmay cause the cessation of one or more element's delivery to a targetcell, and or enable delivery from other sources.

In another configuration, one or more elements dynamically respond tonatural environmental conditions and manifest special functions. Thevarious control laws that regulate biochemical reactions andphysiological processes often display features that allow biomoleculesor biological structures to perform more tasks than are reasonablyexpected from a simple mechanical device. In one embodiment, theinvention takes deliberate advantage of these biological control laws.Via the use of bio- and genetic engineering methods known in the art,the invention makes use of these control laws to dynamically regulatecomplex in vivo and in vitro biochemical reactions and physiologicalprocesses. An example of biological control laws at work is theautomatic self-directed, self-assembly of in vitro and in vivo Clathrinand Coatomer proteins.

In one embodiment, intramolecular dynamics of biomolecules and theconcerted and interlocking steps of conformational changes lead todeliberately purposeful actions. For example, one or more elements mayfit spatially and each step in a process fits temporally (kinetically)with an element of anticipation of the purposeful outcome.

In another example case, the spatially and temporally defined eventsbetween the cell and one or more elements may cause the invention torelease diagnostic and monitoring agents to determine the mostappropriate course of therapeutic action. The calculated utilization ofbiological control laws by one or more elements may, for example,provide for a sophisticated drug delivery system that provides optimaldosing by altering its drug delivery behavior, as well as producingminimal side effect profiles.

A further advantage of the invention is that it provides elements thatcan be bio-engineered to prevent in vivo uptake by one or more types oforgans, tissue, cells, and bone. In the converse, another advantage isthat one or more elements can be bio-engineered for highly selectiveuptake by one or more types of targeted cells, tissue, organs, bone, aswell as by other organic and inorganic matter. In another embodiment,one or more elements comprise a non-selective uptake, non-targeted drugdelivery system.

In another embodiment, the invention provides for the ability of one ormore elements to intelligently monitor, control and regulate, react, andfurther adjust biological processes after delivery of the payload,enabling high precision dosing.

Another advantage of the invention is that Clathrin can cross cellmembranes including the blood brain barrier (Gragera et al 1993) and canmove through the synaptic clefts (Granseth et al 2007). In oneembodiment, bioengineered Clathrin actively transports substances in andout of cells including neurons and blood brain barrier cells.

In another embodiment, one or more elements, operating alone or with oneor more additional elements, comprise one or more types of cell membranecrossing elements and gain access to the cytosol and intracellularelements of one or more types, including one or more cell organelles.Such elements may, in one embodiment, require minimal functionalizationto cross the cell membrane and or enter a cell organelle.

In one embodiment, one or more elements, in whole or in part, in one ormore combinations, take one or more actions to create, spawn, comprise,modify, regenerate, reassemble, and or control and regulate one or morecells, cellular elements and or cellular processes of one or more types.

In one embodiment, one or more elements, in whole or in part, in one ormore combinations, take one or more actions to rectify and or repairfailures and defects in cellular processes, such as, endocytosis,exocytosis, mitosis, trafficking and signaling, and the like. Suchfailures and defects can lead to diseases, for example, cancer.

In one embodiment, one or more elements comprise in situ in vivoelements for remediation, removal and or sequestration of one or moretypes of contaminants, toxins, undesired organic or inorganic elements,and the like.

In one embodiment, one or more elements comprise in situ environmentalelements for remediation, removal and or sequestration of one or moretypes of in vitro environmental contaminants and or toxins; for example,chlorinated solvents TCE, PCE, PCBs, c-DCE, DNAPL, heavy metals(chromium), biofilm, synthetic chemicals, and the like.

In one embodiment, some or all elements may also operate under thecontrol and influence of other in vitro and or in vivo elements,including non-invention elements, and altogether may comprise ascalable, nanoscale platform.

In general, in another aspect, the invention is directed to a method offorming one or more types of scalable platforms, including the steps ofproviding one or more embodiments of the elements to deliberately carryout a series of tasks of one or more types, which tasks and or methodsmay be externally directed or internally self-directed, or a combinationthereof. In other embodiments, one or more nanoscale platforms may beadditionally comprised of one or more non-invention elements andplatforms of one or more types.

One or more elements, in one platform embodiment, may also modify,process, manipulate, encode and decode, input, output, transmit,communicate, store and or read information using techniques and methodsknown in the art, in vivo and in vitro.

In one embodiment, scalable information processing platforms use some orall elements as bits that are programmable into a plurality of logicalstates. In another configuration, the invention features a scalableinformation-processing platform that may include one or more elements.

As a general characteristic, one or more elements may take any suitableform, and multiple embodiments may be used as elements, and or furthercombined in any suitable manner to create one or more cargo carrying andor non-cargo carrying nanoscale elements (“elements”), and ormultifunction nanoscale platforms (“platforms”) of one or more types,operating in vitro and or in vivo, such as: multiple polypeptideelements and platforms; biological elements and platforms; largemolecule elements and platforms; small molecule elements and platforms;biomedical elements and platforms; medical elements and platforms;diagnosis, cure, mitigation, treatment, prevention of disease or othertype of drug elements and platforms; targeted and or non-targeteddelivery elements and platforms; cell, cell organelles, or cell materialcrossing elements and platforms; personal medicine elements andplatforms; elements and platforms that, post administration, in whole orin part enter the central nervous system, including passing the bloodbrain barrier in 30 minutes or less and or in 30 minutes or more;healthcare elements and platforms; reproductive health elements andplatforms; substance abuse disorder treatment elements and platform;bioengineered elements and platforms; cosmetic elements and platforms;agricultural elements and platforms; sensor elements and platforms;research and development elements and platforms; scientific elements andplatforms; crystal elements and platforms; electronic elements andplatforms; photonic energy elements and platforms; informationprocessing or storage elements and platforms; energy storage elementsand platforms; in situ elements and platforms for remediation, removaland or sequestration of undesirable elements and platforms of one ormore types; quantum mechanical elements and platforms; telecommunicationelements and platforms; and the like; one or more of which nanoscaleelements and platforms may be additionally comprised of one or morenon-invention elements and platforms of one or more types, and with orwithout one or more types of cargo elements located on and or in one orall or a subset of elements.

In general, in a further aspect, the invention is directed to a methodof forming one or more formations of nanoscale elements formed in vitrofrom one or more elements of one or more types formed from isolated,synthetic and or recombinant amino acid residues comprising in whole orin part one or more types of Clathrin and or Coatomer I/II proteins ofone or more isoforms, including cloned isoforms; with or without one ormore additional elements of one or more types located on and or in oneor more elements; forming in whole or in part one or more types ofelement carrying and or non-element carrying nanoscale elements andstructures; one or more of which elements may also comprise one or morenon-invention elements of one or more types, forming hybrid elements;wherein one or more elements, using one or more types of methods,executes one or more functions and or effects one or more ends in vivoand or in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects of the invention may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which like reference numbers indicate likeparts.

FIG. 1 is a conceptual diagram depicting a Clathrin triskelion comprisedof one or more elements of one or more types employed in an illustrativeembodiment of the invention.

FIG. 2 is a conceptual cross-sectional view of one or more Clathrinprotein, receptor, adaptor protein, and cargo elements in anillustrative embodiment.

FIG. 3 is a computer generated frontal view of an actual Clathrin cagecomprised of a plurality of Clathrin triskelia, and, in an illustrativeembodiment, comprising one or more invention elements.

FIG. 4 is a flow diagram depicting conceptually the formation ofindividual Clathrin elements during endocytosis, which also serves toillustrate how the instant invention operates in one or moreembodiments.

FIG. 5 is a conceptual diagram depicting Coatomer I/II protein comprisedof one or more subunit and domain elements of the type employed in anillustrative embodiment of the invention.

FIG. 6 is an exemplary energy level diagram 600 illustrating the energylevels associated with a hyperfine interaction between electron andnuclear spin in the presence of magnetic fields.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The instant invention is comprised of one or more formations ofnanoscale elements formed in vitro from one or more elements of one ormore types formed from isolated, synthetic and or recombinant amino acidresidues comprising in whole or in part one or more types of Clathrinand or Coatomer I/II proteins of one or more isoforms, including clonedisoforms, and which operate in vitro and or in vivo. In one embodiment,one or more elements form one or more configurations of one or moretypes, described below.

FIG. 1 is a conceptual diagram illustrating the basic unit of Clathrin,a three-leg pinwheel protein structure, and each complete leg istypically called a ‘monomer’. The arrangement of the monomers in thethree-dimensional protein is the quaternary structure. Each Clathrin legmonomer is further comprised of two subunits, one 190 kDa subunit(“heavy chain”) and one 24-27 kDa subunit (“light chain”). Three,two-subunit Clathrin monomers self-assemble and combine to createtriskelion element 100. It is this triskelion morphology that allowsClathrin to form its unique polyhedral network.

In FIG. 1, the assembled triskelion element 100 is comprised of threemonomer leg elements 102 a-102 c. The three leg elements 102 a-102 cextend radially from a hub section 108. The filamentous portion ofClathrin triskelion legs 102 a-102 c is formed by a continuoussuperhelix. A naturally occurring Clathrin leg is about 47.5 nm (475 Å)long. In the instant invention, Clathrin leg length and or molecularweights can be modified and or adjusted by using bioengineeringtechniques known in the art.

In the case of humans, there are two isoforms each of Clathrin heavychain (CHC17 and CHC22) and light chain (LCa and LCb) subunits, allencoded by separate genes. CHC17 forms the ubiquitous Clathrin-coatedvesicles that mediate membrane traffic. CHC22 is implicated inspecialized membrane organization in skeletal muscle. CHC17 is bound andregulated by LCa and LCb, whereas CHC22 does not functionally interactwith either light chain.

In one embodiment, a Clathrin triskelion is composed of a trimer ofheavy chains 104 a-104 c each bound to a single light chain 106 a-106 c,respectively. In the case of one isoform embodiment, CHC17, a Clathrinheavy chain element is comprised of a 1675 amino acid residue protein,which is encoded by a gene consisting of 32 exons. In the case ofanother isoform embodiment, CHC22, a Clathrin heavy chain element iscomprised of a 1640 amino acid residue protein.

In one or more invention embodiments, efficacious elements formed inpart from Clathrin amino acid residues include, but are not limited to,a N-terminal globular domain 110 a-110 c (residues 1-494) that interactswith adaptor proteins (e.g., AP-1, AP-2, b-arrestin), a lightchain-binding region (residues 1074-1552), and a trimerization domain(residues 1550-1600) near the C-terminus.

One or more of the Clathrin heavy chain amino acid sequences asdescribed in Tables 1 and 2, but not limited to, and in whole or in partmay be modified, altered, adapted or functionalized in one or more waysin one or more embodiments of the invention.

In the illustration, the three Clathrin monomer elements 102 a-102 c arecomprised of six subunit elements, three of which subunits are the heavychain subunit elements 104 a-104 c. The three heavy chain subunits arecomprised of several distinct domains and segments, one or more of whichmay comprise one or more invention elements in one or more embodiments,and may be functionalized via one or more techniques known in the art.

In general, each heavy chain comprises eight repeated motifs (CHCR 0-7),which make up the proximal, knee, distal and ankle segments of aClathrin leg. The heavy-chain amino terminus folds into the terminaldomain (TD) and is attached to CHCR0 by a helical linker. (Brodsky,2004). The three Clathrin heavy chains are joined at their C-termini(located within hub element 108), extending into proximal and distal legdomains ending in globular N-terminal domain elements 110 a-110 c, andwhich are responsible for peptide binding. The Clathrin heavy chainterminal domains provide multiple interaction sites for a variety ofadaptor proteins (AP) that can bind multiple receptors occupied byligands. These sites prevent chemical interactions between cargoelements. The heavy chain N-terminal domain elements 110 a-110 c areeach comprised of a seven-bladed beta-propeller connected to a flexiblelinker region, respectively. This propeller domain interacts with a hostof accessory proteins participating in receptor-mediated endocytosissuch as adaptor proteins, non-visual arrestins and the uncoating ATPase,hsc70. The propeller domain is followed by a long filamentous segment,which is interrupted by a bent region between the distal and proximaldomains, and ends in the trimerization domain at the C-terminus.

Besides harboring determinants important for driving the association ofindividual Clathrin molecules during lattice formation, each of thethree heavy chain 104 a-104 c proximal domains also include bindingsites for attaching the three light chain subunit elements 106 a-106 c,respectively, forming three complete Clathrin monomers. The three lightchain subunits are also comprised of several distinct domains andsegments, one or more of which may comprise one or more inventionelements in one or more embodiments, and may be functionalized via oneor more techniques known in the art.

Among other roles, Clathrin light chains prevent Clathrin heavy chainsfrom interacting with each other. On the other hand, assembly proteinsbind to light chains and cause a change in them such that they no longerprevent heavy chains from interacting. Clathrin light chains consist ofwhat has been described as a linear array of domains: regions of proteindiscernable from the primary sequence or with distinct biochemicalproperties. These are an N-terminal segment, a region that is 100%conserved between light chains, a portion to which Hsc70 binds, acalcium binding domain, a region which binds the heavy chain, a site forneuronal-specific splice inserts and then finally a calmodulin-bindingdomain at the C-terminus domain (Royle, 2006). The light chainC-terminal residues are also important for enhancing the in vitroassembly of hub 108 at low pH.

One or more of the Clathrin light chain amino acid sequences asdescribed in Tables 3 and 4, but not limited to, and in whole or in partmay be modified, altered, adapted or functionalized in one or more waysin one or more embodiments of the invention.

In one embodiment, each of the 3 heavy chain subunits 104 a-104 c mayeach have 3 light chains subunits 106 a-106 c attached, respectively,forming the typical, three-monomer Clathrin triskelion structure. But inanother embodiment, each leg 102 a-102 c may include only the 3 Clathrinheavy chain subunits 104 a-104 c, respectively, which is distinctlyunique from the classic Clathrin monomer configuration. In yet anotherunique embodiment, only 3, non-attached light chain subunits 106 a-106 care used.

In one distinctive embodiment of the invention, a 3-legged pinwheelconfiguration 100 is not enabled, and only partial pinwheel structuresare used. In one embodiment, a partial pinwheel configuration of one ortwo legs (one or two Clathrin monomers) is comprised of one or twoClathrin heavy chains and one or two corresponding light chain subunits.In another embodiment, one or two elements comprised of only one or twoClathrin heavy chain subunits are used; e.g., subunits 102 a, or 102a-102 b. In one embodiment, only one or two unattached light chainsubunits are used.

In another distinctive embodiment of the invention, one or more elementsof one or more types are formed from isolated, synthetic and orrecombinant amino acid residues comprising in part one or more types ofClathrin heavy chain and or light chain proteins of one or more isoformsas described in Tables 1-4.

In one embodiment, one or more N-terminal domain elements, e.g., 110 a,110 b and or 110 c are bioengineered to facilitate, modify, regulate orcontrol peptide binding of one or more types, as well as interactionsites for one or more types of adaptor proteins.

In one embodiment, one or more domain elements of heavy chain subunitsand or light chain subunits are bioengineered to facilitate, modify,regulate or control one or more Clathrin protein characteristics and orbehaviors in vivo and or in vitro.

FIG. 2 is a conceptual cross-sectional view of a biological endohedralconsisting of Clathrin protein elements. In this illustrativeembodiment, one or more elements 102 a-102 c, 106 a-106 c, 104 a-104 c,110 a-110 c, element 108, and or one or more types of elements formedfrom isolated, synthetic and or recombinant amino acid residuescomprising in whole or in part one or more Clathrin proteins of one ormore isoforms, and with or without one or more additional elements ofone or more types, may comprise one or more multiple polypeptideelements of one more types. The latter are labeled in FIG. 2 as elements206 a, 204 a, 202 a, and 208 a, which are formed in vitro, and also mayoperate in vitro and or in vivo. One or more of elements 206 a, 204 a,202 a, and or 208 a may comprise one or more types of functionalization,include invention and non-invention elements, express one or more typesof functionality, and or form one or more types of structures.

In one illustrative embodiment, but not limited to, one or more elements206 a may comprise one or more elements 102 a-102 c, 106 a-106 c, 104a-104 c, 110 a-110 c, element 108, and or one or more types of elementsformed from isolated, synthetic and or recombinant amino acid residuescomprising in whole or in part one or more Clathrin proteins of one ormore isoforms, and express one or more types of functionality in one ormore embodiments.

In another embodiment, one or more elements 206 a may be comprised of,and or help comprise one or more types of non-invention elements, suchas a natural cell element in one embodiment, comprising one or moretypes of hybrid elements in one or more embodiments.

In another embodiment, one or more elements 206 a may be comprised of,and or help comprise one or more types of isolated, synthetic,recombinant and or natural molecules in one or more embodiments.

In one illustrative embodiment, but not limited to, one or more elements202 a may comprise cargo elements of one or more types, includingnatural, isolated, synthetic and or recombinant, including natural andor synthetic ligands and or drugs, and may express more than one type offunctionality. In one embodiment, one or more other elements, of one ormore types, including invention and non-invention elements each may bondwith one or more respective cargo elements 202 a.

In one embodiment, one or more cargo elements 202 a are cavity formingand are non-permeable, semi-permeable, and or permeable, and or canchange from one permeable state to another. In one embodiment, thecavity forming elements comprise one or more types of elements and oragents, including gas, vapor or fluid, with or without dopants. In oneembodiment, one or more cargo cavities elements comprise one or moretypes of elements and or agents, including one or more types of metals.

In another illustrative embodiment, one or more efficacious cargoelements 202 a carried on one or more elements may comprise the totalfunctionality. In another embodiment, one or more other elements, of oneor more types, including invention and non-invention elements may act inconcert with one or more cargo elements 202 a to achieve ensembleefficacy.

In one embodiment, but not limited to, one or more elements 204 a maycomprise attachment and or receptor elements for one or more elements202 a of one or more type, and or express more than one type offunctionality. In one embodiment, one or more other elements, of one ormore types, including invention and non-invention elements each may bondwith one or more respective elements 204 a. In another embodiment,receptor molecules 204 a can be bioengineered to recognize and associatewith specific molecules, which may also be synthetic and or naturalligands and or drugs. In another embodiment, receptor molecules 204 acan be natural, isolated, synthetic and or recombinant.

In one embodiment, but not limited to, one or more elements 208 a of theinstant invention may comprise the major types of adaptor elements, likethe heterotetrameric adaptor protein (AP) elements, and the monomericGGA (Golgi-localizing, Gamma-adaptin ear domain homology, ARF-bindingproteins) adaptors. In one illustrative embodiment, elements 208 acomprise one or more small sigma subunits of various adaptins fromdifferent AP adaptor elements. The AP complex family has six members inmammals: AP-1A, AP-2, AP-3A and AP-4 are ubiquitously expressed. Theother two members, AP-5 and AP-6, are cell-type specific isoforms ofAP-1A and AP-3A: the epithelium-specific AP-1B and the neuron-restrictedAP-3B. (Ohno, 2006). In another embodiment, AP180, like AP-2 and AP-3,binds to N-terminal domains 110 a-110 c of Clathrin. In one embodiment,one or more AP elements may be functionalized at one or more heavy chainterminal domain elements 110 a-110 c. In one embodiment, one or moreother elements, of one or more types, including invention andnon-invention elements each may bond with one or more respectiveelements 208 a. In another embodiment, adapter molecules 208 a arebioengineered to recognize specific receptor molecules and to couple thereceptor molecules to Clathrin and or Coatomer protein elements. Inanother embodiment, adapter molecules 208 a can be natural, isolated,synthetic and or recombinant.

In one embodiment, one or more elements 206 a, 204 a, and or 208 aoperate alone without cargo element 202 a, and comprise one or moretypes of inherently efficacious solo acting elements.

In one embodiment, unlike prior Clathrin art, a plurality of elements206 a, 204 a, and or 208 a operate without cargo elements 202 a, andcomprise an inherently efficacious cage element 212 of one or moretypes, like a drug element, for example, which is unlike prior Clathrinart.

In one embodiment, also unlike prior Clathrin art, a plurality ofelements 206 a, with or without one or more additional other elementscomprise cage element 212, and element 212 has one or more elements, ofone or more types and affixed via one or methods, located on the outsidepart of cage element 212; that is, located outside the cavity formed bycage 212. In another embodiment, further unlike prior Clathrin art, aplurality of elements 206 a, with or without one or more additionalother elements, comprise cage element 212, and element 212 has one ormore elements, of one or more types and affixed via one or methods,located on both the outside, and inside parts (i.e., located within thecage cavity), of cage element 212.

According to one invention feature, cargo attachment element 204 a andor element 208 a shields cargo element 202 a in the same element 206 afrom interacting. According to another feature, the shielding propertiesof element 206 a shields and inhibits chemical and molecularinteractions between it and the external environment. According to afurther feature, element 206 a protectively sequesters cargo elements202 a from the external environment.

In another embodiment, one or more non-invention, “natural” Clathrinelements 206 b-206 f (the term “natural” hereinafter generally refers tonon-isolated, non-recombinant, and non-synthetic protein elements) joinwith one or more isolated, recombinant, and or synthetic elements; inthis example, 206 a; to form a natural/invention hybrid Clathrin cageelement 212. In another embodiment, hybrid cage element 212 may also becomprised of natural cage element 220, which is a vesicle, forming ahybrid Clathrin Coated Vesicle.

FIG. 3 is a computer generated frontal view of a Clathrin cage 300comprised of a plurality of natural Clathrin triskelia elements 302-308,respectively. In an illustrative embodiment, element 310 is an inventionelement, comprised of three heavy chain elements 104 a-104 c—which mayor may not include three respective light chain elements 106 a-106c—forming a hybrid or fused cage 300 comprised of natural elements andinvention elements. In this role, element 310 comprises an efficaciousreplacement for a natural triskelia element.

FIG. 4 is a flow diagram 400 depicting, conceptually, the formation of aplurality of natural Clathrin elements 206 b-2026 f, and, in thisexample, along with invention element (206 a) into cage 200, which atstep 440, shows Clathrin coated vesicle 220. The process by whichnatural Clathrin molecules 206 b-206 d obtain natural cargo molecules202 b, 202 c, and 202 d in this example is known as Clathrin mediatedendocytosis (CME), a process wherein a cell takes in macromolecules byforming vesicles derived from the plasma membrane. Endocytosis iscrucial to cellular function. Via CME, cells internalize cargoattachment elements, transmembrane channels, transporters andextracellular ligands such as hormones, growth factors and nutrients.

In one embodiment, one or more invention elements are biologicallyengineered to take or induce one or more types of actions, such as tocreate, spawn, comprise, modify, repair, regenerate, reassemble, and orcontrol and regulate CME, as well as exocytosis, mitosis, trafficking,signaling processes, other behaviors, and the like. Defects anddisorders in any of these critical cellular processes can lead todisease, and one or more types of these processes may be modified in oneor more embodiments of the instant invention, for example, to achievetherapeutic effect.

In one embodiment, the instant invention takes or induces one or moreefficacious actions involving receptor-mediated endocytosis thatencompass nutrient uptake (LDL, transferrin, etc.), membrane recycling,membrane protein recycling, antigen uptake, synaptic vesicle recycling,and signaling receptor down-regulation.

In one or more embodiments, one or more invention elements comprisecounterparts to natural Clathrin proteins that may inherently behave asa drug; e.g., one or more invention elements are functionalized for invivo delivery and carry no additional elements, such as cargo. Such soloacting element embodiments would interact in one or more ways withnatural cells and their processes, and by so doing diagnose, regulateand or cure one or more diseases and disorders relating to endocytosis.

An increase of a cellular component is called upregulation. Upregulationis an increase in the number of receptors, e.g., see elements 204 b, 204c, and 204 d in FIG. 4, on the surface of target cells, making the cellsmore sensitive to a hormone or another agent. For example, there is anincrease in uterine oxytocin receptors in the third trimester ofpregnancy, promoting the contraction of the smooth muscle of the uterus.In one or more embodiments, one or more invention elements, either byacting alone and or in part with other elements of one or more types,including natural and or non-invention elements, efficaciously modify,control and regulate, interfere with, create, and or spawn elements, andor induce actions or behaviors that increase the upregulation of one ormore types of receptors of the surfaces of target cells.

On the other hand there is downregulation, an example of which is thecellular decrease in the number of receptors to a molecule, such as ahormone or neurotransmitter, which reduces the cell's sensitivity to themolecule. In the literature, downregulation is the process by which acell decreases the quantity of a cellular component, such as RNA orprotein, in response to an external variable. In one or moreembodiments, one or more invention elements, either by acting alone andor in part with other elements of one or more types, including naturaland or non-invention elements, efficaciously modify, control andregulate, interfere with, create, and or spawn elements, and or induceactions or behaviors that increase the downregulation of one or moretypes of receptors.

Exocytosis is the reverse process of endocytosis, whereby a cell directssecretory vesicles out of the cell membrane. These membrane-boundvesicles contain soluble proteins to be secreted to the extracellularenvironment as well as membrane proteins and lipids that are sent tobecome components of the cell membrane. Exocytotic vesicles are usuallynot Clathrin-coated; most of them have no coat at all. However, twoobservations suggest that Clathrin effectively ‘tracks’ vesicle proteinsleaving a synapse. In one study (Granseth, et al, 2008) the amount of aClathrin light chain (LC) tagged with the element mRFP leaving thesynapse was proportional to the number of vesicles released by thestimulus, as assessed by the amplitude of a sypHy signal (sypHy is animproved fluorescent reporter of exocytosis). Second, in the same studythe movement of LC-mRFP began without a significant delay and peakedwith the sypHy signal. The movement of Clathrin out of the synapsetogether with synaptophysin and synaptobrevin is most easily explainedas representing CME (Clathrin mediated endocytosis) of vesicles at sitesremoved from the active zone. This interpretation is consistent withstudies showing that the machinery for CME is not at the active zone,but in the surrounding regions of membrane (Heuser & Reese, 1973;Ringstad et al. 1999; Qualmann et al. 2000; Teng & Wilkinson, 2000).Thus, Clathrin is naturally found in the extracellular space and mayplay a role in regulating exocytosis and or endocytosis. In one or moreillustrative embodiment, one or more elements of one or more types mayefficaciously operate in inter- and or extra-cellular spaces of one ormore types; for example, perform remediation, sequestration, or removalof one or more types of undesirable elements.

Membrane trafficking only occurs during interphase. As the cell entersmitosis, Clathrin-mediated membrane traffic is rapidly shut down andonly resumes in late telophase. Clathrin may therefore have a separatefunction that is distinct from membrane trafficking, which operatesduring mitosis. Clathrin is thus a multifunction protein: duringinterphase its function is in membrane trafficking and during mitosis ithas a role in stabilizing spindle fibers (Royle, 2006). In one inventionembodiment, mitosis may be efficaciously controlled and regulated,modified, and or induced via one or more methods and instances of theinstant invention.

In another embodiment, one or more elements are comprised of, but notlimited to, one or more isolated, synthetic, and or recombinant adaptorprotein molecules, tubulin protein molecules, dynamin protein molecules,epsin protein molecules, endophilin protein molecules, synaptotagminprotein molecules, and or other types of protein molecules associatedwith Clathrin and Coatomer proteins and processes, for efficaciouseffect.

In another embodiment, one or more natural adaptor protein molecules,tubulin protein molecules, dynamin protein molecules, epsin proteinmolecules, endophilin protein molecules, synaptotagmin proteinmolecules, and or other types of protein molecules involved withassociated with Clathrin and Coatomer proteins and processes formefficacious hybrid elements when also comprised of one or more types ofinvention elements.

The CME process involves a dynamic interaction between Clathrin and awide range of other protein molecules, and altering the compositions andbehaviors of the various molecular parties involved. For example, thecell uses endocytosis to control and regulate the density of receptorson the cell surface and to acquire nutrients. Endocytosis ofligand-activated cargo attachment elements is essential for the properattenuation of a variety of signal transduction processes, as well asfor co-localization of activated cargo attachment elements withdownstream signaling molecules. Endocytosis also counterbalancessecretion, preventing continuous expansion of the plasma membrane.Endocytosis thus internalizes macromolecules and fluid, and aftersorting, directs the internalized molecules for degradation orrecycling.

The endocytosis process begins when proteins bound to cargo attachmentelements accumulate in coated pits 404, which are specialized regions ofthe cell membrane 402 where it is indented and coated on its cytoplasmicside with a bristle-like coat composed of two natural proteins: Clathrinand protein adapters. Most, if not all, intracellular transport vesiclesare encased in a proteinaceous coat, one class of which isClathrin-coated vesicles (CCVs). CCVs also mediate the transport oflysosomal hydrolases from the trans-Golgi network, as well as theefficient internalization of extracellular solutes such as nutrients,hormones, growth factors, and immunoglobulins at the plasma membrane.

Clathrin also transports proteins from the Golgi to other organelles. Inneurons, endocytosis is critical to allow rapid synaptic vesicleregeneration. Besides Clathrin, there are other coat-forming proteins,such as COP I and COP II, which mediate intracellular traffic and thereare Clathrin-independent endocytic pathways which mediateinternalisation of a variety of cargo (Royle, 2006).

In one invention embodiment, the natural endocytosis process istransformed into a versatile therapeutic method to regulate theintensity, localization, half-life and function of signaling elements(signalosomes) that form in cells upon, for example, binding of growthfactors, cytokines and morphogens to their cognate receptors. In oneexample embodiment, the invention rectifies breakdowns in the functionof endocytic adaptors that might facilitate impairment of tissuehomeostasis and consequent tumor development. In another illustrativeembodiment, one or more invention elements, acting alone or not,interact with natural adaptor proteins required for appropriate receptordownregulation and which play distinct roles in oncogenesis. (Crosetto,et al. 2005) In another embodiment, CME elements might also comprise oneor more invention cargo elements (202 a in FIG. 4), which can be drugs,other ligands, and the like.

In one embodiment, referring to FIG. 4, a natural Clathrin coatedvesicle 220 is desired to form to endocytose over-expressed naturalreceptor elements 204 b and 204 c that are initially located outsidecell membrane 402. The appearance of one or more types of inventionelements, such as element (206 a) in the illustrative example, outsidecell membrane 402 and or by crossing 402, dynamically begin to create,induce, spawn, mediate, control and regulate, regenerate, and orinteract with one or more natural endocytosis processes and behaviors.With the prompting of one or more types of invention Clathrin elements,one or more biological processes acting on cell membrane 402 induce aClathrin bud 404 to form at 420.

As shown at 430 and 440, after forming completely around bud 404,natural Clathrin elements 206 b-206 d pinch off (scission) from membrane402 with the desired over expressed receptors 204 b and 204 c heldinside vesicle 220. After excision, bud 404 has evolved into a pluralityof natural Clathrin elements 206 b-206 f, some of which are attached toone or more types of over expressed receptor elements 204 b and 204 c,as well as attached to other receptor elements; which in this exampleare the normally expressed natural elements 204 d.

In one illustrative embodiment, the otherwise all-natural plurality ofClathrin elements in FIG. 4 includes one or more non-cargo carrying;solo acting invention elements (206 a), forming a “hybrid” CCV 440 withthe desired efficacious properties and behavior. This hybrid CCV thenfollows normal pathways within the cell, causing downregulation of thedesired over-expressed receptor elements, which may be associated withone or more types of neurotransmitters, viruses, cholesterol, as well aswith other cargo types, restoring a cell to its normal, healthy state.

In another illustrative embodiment, natural Clathrin coated vesiclestructure 440 in FIG. 4 is additionally comprised of one or morenon-cargo carrying invention receptor element 204 a and or adaptorelement 208 a (as illustrated in FIG. 2), forming a hybrid or fusedClathrin coated vesicle 440 in FIG. 4, with the desired efficaciousproperties and behavior. In another embodiment, one or more hybridizedand or invention elements may enter the cell nucleus and or otherorganelles and cell elements.

The fusion and or participatory actions of one or more non-additionalelement carrying, solo acting invention elements 206 a, 204 a, and or208 a in FIG. 2 may yield a therapeutic effect, and are an exampleembodiment of inherently efficacious invention elements in action. Inanother embodiment, natural or hybrid CCV 440 in FIG. 4 also includesone or more invention cargo molecules (202 a) that may have beentransported into the cell via their attachment to one or more naturaland or invention receptor elements.

Referring again to FIG. 4, in another example embodiment, a therapeuticeffect is accomplished via one or more invention elements by regulatingEGFR (epidermal growth factor receptor), which exists on the cellsurface and is activated by binding of its specific ligands includingepidermal growth factor and transforming growth factor a (TGFa). Whenthese natural cargo attachment elements are activated, cells rapidlyclear them from the surface and destroy them. Control of EGF receptorsignaling is performed by Clathrin-mediated endocytosis. NaturalClathrin coats also exist on endosomes and are involved in endosomalsorting of the EGFR. A defect in this overall process will likely leadto uninhibited growth of cells and tumors. EGFR expression,over-expression, or mutation is associated with cancer progression,advanced disease, drug resistance, aggressive disease, poor prognosis,and reduced survival. EGFR is considered one of the main proteinselevated in breast, lung, and prostrate cancers, among others. Braincancer is also implicated with over-expressed EGFR. Other work has shownthat using monoclonal antibodies for EGFR, or anti-EGFR, has proven aneffective strategy for getting nanoparticles to specifically attachthemselves to cancer cells. Additional work has shown effectiveness ofEGFR as the cancer-targeting pathway. In one embodiment, CME, cellfusion, cell penetrating, and or one or more types of otherparticipatory actions of one or more solo operating, efficaciousinvention elements 206 a, 204 a, and or 208 a in FIG. 2 may yield atherapeutic effect in controlling, regulating, or mediating EGFRactivity. In another example embodiment of modulating EGFR activity,cargo elements (202 a) in FIG. 4 may comprise one or one or more typesof cancer drugs or biologicals delivered directly into cells andorganelles that are transported into the cell via their attachment toone or more natural and or invention receptor elements during CME, bycell fusion, by directly penetrating cell membrane 402, and or by one ormore types of other participatory actions. In another embodiment,invention cargo elements (202 a) may comprise one or more diagnosticagents, or combine one or more diagnostic agents and therapeutic agentsin the same payload. In one or more embodiments, one or more inventionelements of one or more types may thus comprise an efficacious methodfor the diagnosis, treatment, remedying, curing, and or prevention ofone or more types of cancers, including those cancer types that falloutside the scope of EGFR-related activity.

FIG. 5 is a conceptual diagram illustrating the basic units of CoatomerI and II proteins. COPII and Clathrin cages are both constructed from∂-solenoid and ß-propeller building blocks (Fotin et al., 2004b; terHaar et al., 1998; Ybe et al., 1999). In various embodiments of theinvention, one or more elements of one or more types are formed fromisolated, synthetic and or recombinant amino acid residues comprising inwhole or in part one or more types of Coatomer proteins of one or moreisoforms, including cloned isoforms. Examples of various Coatomersubunit amino sequences are listed in Tables 5-11. In anotherembodiment, one or more Coatomer subunit amino acid sequences may bemodified, altered, adapted or functionalized in one or more ways in oneor more embodiments of the invention.

In one embodiment, Coatomer is comprised of seven distinct subunits:alpha, beta, beta′, gamma, delta, epsilon and zeta subunits,respectively.

In Clathrin, a triskelion assembly unit lies at each vertex, and the∂-solenoid legs of neighboring triskelia interdigitate extensively asthey extend toward the adjacent vertices; the ß-propeller is not part ofthe architectural core and instead projects in toward the membrane tointeract with adaptor molecules (Fotin et al., 2004; Kirchhausen, 2000).In contrast, the COPII assembly unit is a rod that constitutes the edgeof a cuboctahedron, and four rods converge to form the vertex with nointerdigitation of assembly units. ∂-solenoid domains form the core ofthe edge, but, unlike Clathrin, the COPII vertices are formed fromß-propellers. In summary, the COPII and Clathrin lattices seem not toshare common construction principles other than the use of ∂-solenoidand ß-propeller folds.

Crystallographic analysis of the Coatomer II assembly unit reveals a 28nm long rod, element 502, comprising a central solenoid dimer capped bytwo B propeller domains, elements 504, at each end. GTPase, elements508, bind to adaptor elements 506, which bind to elements 502. In theillustration, element 502 a is an invention element that acts as anefficacious replacement element for one or more natural element 502,forming a hybrid Coatomer element. The structural geometry andproperties of COPI coats remain to be determined. However, by analogy tothe COPII and Clathrin structural units, they probably involve apreassembled cage protein (CP) scaffold that is generated by theß-propeller-containing and ∂-solenoid-containing subunits and an adaptorprotein (AP) subcomplex. Together these could form an AP-CPheptaheteromeric functional unit in the cytosol. (Gurka, et al. 2006)

COPI and COPII play a major role in exocytosis, as also can theirinvention element counterparts. Clathrin can also play a role inexocytosis, but to a lesser extent than Coatomer. The exocytosis processrefers to the fusion of intracellular vesicles with the plasma membrane.It occurs via two major processes, a constitutive pathway and aregulated pathway. These are the major ways that the cell secretesmaterials, wherein a cell secretes macromolecules (large molecules) byfusion of vesicles with the plasma membrane. Coatomer-coated vesicles,which are typically less than fifty nanometers in size, are alsoinvolved in vesicular transport between the Golgi apparatus, endoplasmicreticulum and plasma membrane. Coatomer I vesicles shuttle elements fromthe Golgi to the endoplasmic reticulum (ER). Coatomer II vesiclesshuttle elements from the ER to the Golgi. Coat-protein I/II subunits(COPs) require ATP to assemble into a coat and unlike Clathrin coats,the Coatomer coat remains on the vesicle until docking occurs. In someinstances, Coatomer proteins are also involved in endocytosis, but areunrelated to Clathrin. Thus, while Clathrin also mediates endocyticprotein transport from the ER to the Golgi, Coatomers (COPI, COPII)primarily mediate intra-Golgi transport, as well as the reverse Golgi toER transport of dilysine-tagged proteins. Coatomers reversibly associatewith Golgi (non-Clathrin-coated) vesicles to mediate protein transportand for budding from Golgi membranes. In one or more embodiments, one ormore COPI/COPII invention elements and or Clathrin invention elements,either by acting alone and or in part with other elements of one or moretypes, including natural and or non-invention elements, efficaciouslymodify, control and regulate, interfere with, create, and or spawnelements and or induce actions or behaviors involving exocytosis.

Cells of the mammalian immune system undergo selective changes inprotein glycosylation during differentiation, immune activation, andautoimmune disease. In many, if not most of these types of diseasesendocytosis and cellular trafficking and signaling plays a role.Referring again to FIGS. 1, 2, 3, 4, (and 5, in some embodiments), butnot limited to, in one embodiment, one or more invention elements of oneor more types, in whole or in part selectively interfere with, fusewith, control and regulate, induce, and otherwise modify endocytosis,receptor-specific processing, trafficking and signaling, and otherbehaviors for efficacious effect in one or more types of autoimmunediseases, including, but not limited to, one or more types of diabetes,CNS autoimmune diseases, and other types of autoimmune diseases thateffect the body.

Referring again to FIGS. 1, 2, 3, 4, (and 5 in some embodiments), butnot limited to, in one embodiment, one or more invention elements of oneor more types selectively interfere with, control and regulate, and ormodify secretory products that participate in inflammation andimmunoregulation; and also in other embodiments, whereby endocytosismediated by specific receptors for immunoglobulin or by other opsoninsis important in removal of damaged self or foreign particles. In anotherembodiment, defects in membrane receptor function, whether inherited oracquired, and the pathogenesis of immune diseases may be remedied,inhibited, mitigated, and or prevented.

Referring again to FIGS. 1, 2, 3, 4, and 5, in one embodiment, but notlimited to, one or more invention elements of one or more typesefficaciously fuse with and or functionally replace one or more naturalelements commonly found in endocytosis, exocytosis, mitosis, traffickingand signaling, and the like, either by acting alone and or in part withother elements of one or more types, including natural and ornon-invention elements.

Referring again to FIGS. 1, 2, 3, 4, and 5, but not limited to, inanother embodiment, one or more invention elements of one or more typesefficaciously cross over into a cell, its elements, and or itsorganelles, such as its nucleus, either by acting alone and or in partwith other elements of one or more types, including natural and ornon-invention elements.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more invention elements efficaciously create,spawn, comprise, modify, repair, regenerate, reassemble, and or controland regulate one or more natural elements commonly found in endocytosis,exocytosis, mitosis, trafficking and signaling, other cellularbehaviors, and the like, either by acting alone and or in part withother elements of one or more types, including natural and ornon-invention elements.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more invention elements efficaciously utilizenatural and or genetically engineered elements to encode components ofthe intracellular sorting machinery that mediate the selectivetrafficking of lipids and proteins in the secretory and endocyticpathways, to efficacious effect.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more invention elements efficaciously utilizegenetic agents and elements, including, but not limited to, proteins;peptides; DNA and DNA variants; RNA and RNA variants such as mRNA, iRNAand siRNA; RNA-induced silencing complex (RISC), other genetic-modifyingagents and methods, and the like.

In another embodiment, but not limited to, one or more inventionelements efficaciously utilize one or more oligonucleotides in antisensetherapy. These antisense DNA drugs work by binding to messenger RNAsfrom disease genes, so that the genetic code in the RNA cannot be read,stopping the production of the disease-causing protein.

In another illustrative embodiment, one or more elements may compriseone or more RNAi (RNA interference) elements and or RNAi variants suchas small interfering RNA molecules (siRNA), but not limited to, that maycollaborate with proteins in the cell and also may form a nanoscaleelement called a RISC (RNA-Induced Silencing Complex). RNAi and or RISCsmay be used to head off a genetic disease before the first symptomappears, based on an analysis of an individual's predisposition tocertain diseases. This methodology is a way of silencing a specificgene, for example, genes that direct cancer cells to proliferate or thatcreate overproduction of proteins that cause rheumatoid arthritis.Basically, RNAi works by scanning RNA templates that may cause a diseaseand cleaving that RNA template, and enzymes then destroying the templatebefore it can complete its actions on the offending DNA. One of the keybarriers to successful RNAi therapy is their finding their way to aspecific site in the body and then the RNAi not degrading rapidly beforeit can do useful work. In one illustrative embodiment, RNAi, siRNA, RISCelements and or other suitable methods may be targeted by an inventionelement such that one or more such RNA elements seek out and destroypotentially harmful genetic elements and or other genetic processes.

As noted in the literature, Clathrin heavy chain is known to be acytosolic protein that functions as a vesicle transporter. However, theClathrin heavy chain exists not only in cytosol but also in cell nuclei.The p53 gene, in which mutations have been found in >50% of humancancers, encodes a protein that plays an important role in preventingtumorigenesis. Clathrin heavy chain expression enhances p53-dependenttransactivation, whereas the reduction of Clathrin heavy chainexpression by RNA interference (RNAi) attenuates its transcriptionalactivity. Moreover, Clathrin heavy chain binds to the p53-responsivepromoter in vivo and stabilizes p53-p300 interaction to promotep53-mediated transcription. Thus, nuclear Clathrin heavy chain isrequired for the transactivation of p53 target genes and plays adistinct role from Clathrin-mediated endocytosis (Enari, et al 2006). Inone embodiment, p53 and or one or more other types of genes, theirdiseases and disorders, and or RNAi related activities may beefficaciously controlled and regulated, mitigated, prevented, and ormodified via one or more embodiments of the instant invention.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more elements, acting alone or not, would achievetherapeutic effect by deliberately controlling and regulating, ormodifying faulty exocytosis and or endocytosis processes that producedisorders and diseases. This is a health critical situation, as the roleof dopamine receptors and transporters; the excitability of dopaminergicneurons; and the regulation of extracellular dopamine levels in thebrain, especially in relation to the diseased state, has proven to beimperative for a further understanding of dopaminergic neurotransmissionas a whole. For example, dopaminergic neurotransmission criticallydepends on exocytotic release and neuronal uptake of dopamine, as wellas on diffusion away from the release site. Once target cells arereached, dopamine can bind to and activate dopamine receptors. Thesubsequent cellular response depends on the type of dopamine receptorthat is activated and the signal transduction mechanisms that arecoupled to these receptors. Disturbances in one or more of theabove-mentioned aspects of dopaminergic transmission could lead tosevere neurological and neuropsychiatric disorders such as Parkinson'sdisease, depression, addiction, schizophrenia, attention deficithyperactivity disorder, restless legs syndrome, Tourette syndrome, andthe like, and in or more invention embodiments, one or more suchdisorders may be efficaciously treated.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more elements, during some operations mayinteract with, for example, an externally applied magnetic field, likeduring NMR. However, since invention protein elements are electricallyneutral, only minimal (e.g., no) structural distortion of the elementsoccurs in the presence of the magnetic field. Therefore, using inventionelements to capture other types of elements, which may be, for example,one or more NMR contrast agents for developmental imaging and diagnosticstudies, and which contrast agents may also be capable of crossingcellular membranes, protects and extends the utility of the invention.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more elements may comprise, for example, one ormore metal ions including, but not limited to, the gadolinium (III)chelate compounds of DTPA, DO3A, DOTA and other variations of theselinear and macrocyclic ligands that act as targeted and or non-targetedcontrast agents.

Direct Gd3+—OH2 chemical bonds, which exchange rapidly with other bulkH₂O molecules, produce the mechanism whereby unpaired electrons on Gd3+relax the proton nuclei of many nearby H₂O molecules. Accordingly, thebehavior of T1 contrast agents, such as those based on gadoliniumrequires good direct contact with tissue water molecules (spin-latticerelaxation mechanism) to be efficient. Thus, it is often preferable tobind them to the external surface of the carrier. (Hooker, et al. 2007)In one embodiment, one or more elements facilitate better contact totissue water because one or more contrast agents of one or more typesare not located in the interior part of a cage (in its cavity), butrather, located on much more exposed non-cage elements of one or moretypes. In one embodiment, one or more cage element 212 has one or morecontrast agents of one or more types located on the outside part of cageelement 212; or on both the inside and outside parts of element 212.

In another illustrative embodiment, one or more imaging or studyelements comprise one or more treated manganese minerals, such asoxides, silicates, and carbonates for imaging and study enhancement.

Besides Gd3 complexes, there is another important class of contrastagents for MRI that is based on polysaccharide coated iron oxideparticles. Their peculiarity stems from the fact that their bloodhalf-life and distribution to different organs of thereticuloendothelial system (RES) depend upon the particle size (Aime, etal 1998). In one embodiment, one or more elements comprise one or moreof a wide range of lanthano-invention labeled derivatives forcustom-designed contrast agents.

In another embodiment, one or more elements comprise one or moretherapeutic agents in addition to one or more imaging contrast anddiagnostic agents.

In another illustrative embodiment, targeted and or non-targeted in vivodelivery of one or more elements are internally and or externallymonitored, directed, activated, deactivated and or regulated, locallyand or at a remote distance by, for example, but not limited to, NMR,ESR, ultrasound, radio transmissions, and or biochemical reactions.

Additionally, in other embodiments, NMR is combined with othertechniques, such as ENDOR, which combines the best aspects of ESR andNMR, to yield high sensitivity and nuclear selectivity, respectively,for in vivo and in vitro studies.

In one embodiment, one or more different sized, paramagnetic coated,quantum dots, and or photonic dots are used as one or more contrastmarkers in magnetic resonance imaging (Mulder, et al., 2009). In otherembodiments, one or more different sized quantum dots, and or photonicdots may be used in positron emission tomography (PET) for in-vivomolecular imaging, or as fluorescent tracers in optical microscopy.

In another configuration, one or more types of elements comprise one ormore radiodiagnostic agents for nuclear medicine.

Referring again to FIG. 2, in further illustrative embodiments,free-floating cargo may be carried in cavity forming cargo elements 202a that comprise a fluid, gas, or vapor; which free-floating cargo, forexample, may be one or more molecular ensembles for enhanced medicalimaging, and which cargo may also be carrying one or more therapeuticagents.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more invention elements comprise one or moretypes of elements in whole or in part, such as one or more drug andpharmacological elements; biological elements; biomedical or medicalelements; and the like, including healthcare elements; bioengineeredelements; cosmetic elements; and the like.

Referring again to FIGS. 1, 2, 3, 4, and 5, but not limited to, in oneembodiment, one or more elements of one or more types comprise targetedand or non-targeted drug delivery elements, including their highprecision dosing, or other forms of healthcare elements for diagnosing,remedying, inhibiting, mitigating, curing, and or preventing one or moretypes of diseases, infections, physical or mental trauma, or other formsof physical and mental afflictions.

Referring again to FIGS. 1, 2, 3, 4, and 5, but not limited to, in oneembodiment, one or more elements comprise an in vitro and or in vivomodel and or system for research study, including a model, method, andor system for the research and development of new drugs, therapies,prosthetics, and drug delivery systems, including an accelerated drugdiscovery process.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more elements, acting alone or not, are utilizedfor studying, discovering, preventing, curing, mitigating, and orhealing one or more types of animal, tree, plant, grain, grass,agricultural, vegetable, and or fungal diseases, disorders,infestations, and or blights.

Referring again to FIGS. 1, 2, 3, 4, and 5, in another embodiment, butnot limited to, one or more elements are used for studying, discovering,designing, and or enabling of genetically engineered elements, forexample, one or more types of genes, cells, and other biologicalelements and products in animals, trees, plants, grains, grasses,agriculture, vegetables and fungi.

In another illustrative embodiment, one or more elements comprise one ormore methods for nourishing and or promoting healthy growth in one ormore types of animals, trees, plants, grains, grasses, agriculture,vegetables and or fungi.

Referring again to FIGS. 2 and 4, in another embodiment, but not limitedto, the heat shock cognate protein, hsc70, and its molecularco-chaperone auxilin, help to regulate the natural endocytosis aftermathof natural CCV uncoating and disassembly. Hsc70 also promotes uncoatingand disassembly of Coatomer I and II vesicles. In cells over-expressingATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo.In one embodiment, bioengineered elements may be used to regulate underor over expression of hsc70 and or auxilin. In one example embodiment,using a monoclonal antibody or other agent type as cargo against hsc70blocks the hsc70-mediated release of invention and or non-inventionClathrin from coated vesicles. In another example embodiment, or moreauxilin elements comprise invention elements.

In one illustrative embodiment, one or more elements are stable withrespect to dissociation, including one or more associated non-inventionelements.

In another illustrative embodiment, disassembly and dissolution of oneor more elements are deliberately inhibited and control and regulated,including one or more associated non-invention elements.

In one illustrative embodiment, one or more elements remain stable for atime certain or estimated time before the onset of dissociation,including one or more associated non-invention elements.

In one illustrative embodiment, dissociation of one or more elements mayoccur in whole or in part, including one or more associatednon-invention elements.

In one illustrative embodiment, one or more cargo elements may compriseone or more uncoating and dissociation agents and or use one or moremethods for controlled and regulated release of agents or cargo from oneor more elements, including one or more associated non-inventionelements.

In another embodiment, disassembly and dissolution of one or moreelements, including one or more associated non-invention elements areinhibited, controlled and regulated, and or promoted by using one ormore specific agents, stimuli, and or other methods.

In one embodiment, but not limited to, one or more invention elements ofone or more types are formed in vitro via the following protocols, whichmay be modified and or substituted by one or more other types ofprotocols in one or more invention embodiments: (Adapted from Campbell,C et al., Biochemistry 23, 4420-4426 (1984), Pearse & Robinson, EMBO J.9:1951-7 (1984), and Zhu, et. al., Methods in Enzymology, 328, 2001,Kedersh N, et al., J. Cell Biology 103, 1986.)

(Adapted from Campbell, C et al., Biochemistry 23, 4420-4426 (1984),Pearse & Robinson, EMBO J. 9:1951-7 (1984), and Zhu, et. al., Methods inEnzymology, 328, 2001, Kedersh N, et al., J. Cell Biology 103, 1986)

Part I. Method of Differential Centrifugation.

-   -   1. Make up 1 L of a buffer (buffer A) that comprises: 50 mM Mes        pH 6.5, 100 mM NaCl, 1 mM EGTA, 0.5 mM MgCl₂, 0.02% NaN₃, 1 mM        DTT a day prior to experiment and storage at 4° C.    -   2. Add 1:100 PMSF proteases inhibitor to buffer A (200 ul/20        ml).    -   3. Collect and wash 14 rat brains (˜2.0 g) and livers (˜20.0 g).        Wash and place the brains in ice-cold buffer A. Perfuse the        livers with ice-cold PBS and collect them in ice-cold buffer A.    -   4. Mince and homogenize the brains in a Potter-Elvehjem grinder        with 2 volume of ice-cold buffer A per total brain wet weight        (˜90 ml). Do the same with the livers (˜400 ml).    -   5. Centrifuge the homogenate at 23,000 g (11,900 rpm) in a        Sorvall GSA or at 13,000 rpm in a Sorvall SS34 rotor for 45 min        at 4° C.    -   6. Collect the supernatant and centrifuge at 43,000 g (18,000        rpm) in a Sorvall SS34 rotor or at 20,000 rpm in a ti 45 Beckman        rotor for 1h at 4° C.    -   7. Resuspend the pellet in 10 ml of ice-cold buffer A, use a        loose-fitting Teflon-glass Dounce homogenizer.    -   8. Collect homogenate in a 50 ml conical tube. Wash pestle and        glass homogenizer with 5 ml of buffer A, and add this to        homogenate until total volume is 15 ml. Add 1:100 PMSF    -   9. Dilute the homogenate 1:1 with 15 ml of 12.5% Ficoll/12.5%        sucrose (both in ice-cold buffer A), and mix by inversion to        ensure homogeneity.    -   10. Centrifuge at 43,000 g (18,000 rpm) in a Sorvall SS34 rotor        or at 20,000 rpm in a ti 45 Beckman rotor for 30 min at 4° C.    -   11. Collect the supernatant in a graduate cylinder and dilute it        1:5 in ice-cold buffer A. Add 1:100 PMSF    -   12. Centrifuge the supernatant at 100,000 g (33,000 rpm) in a        Beckman 70.1Ti rotor or at 31,100 rpm in a ti 45 Beckman rotor        for 1h at 4° C.    -   13. Collect pellet and resuspend in 5-10 ml of ice-cold buffer A        by using a loose-fitting Teflon-glass Dounce homogenizer. Add        1:100 PMSF    -   14. Leave the homogenate on ice for about 30 min, and take an        aliquot of 10 ul for EM, and dilute 1:10 for brain, 1:100 for        liver.        Part II. Purification of CCVs Using Density Gradients (Zhu's        CCVs and Clathrin Coat Preparation). Submit the Crude        Clathrin-Coated Vesicles from Fresh Rat Brain to Discontinuous        Sucrose Gradient for Remove Contaminating Vaults.    -   1. CCVs resuspended in (5-10 ml) buffer A    -   2. Preparer a discontinuous sucrose gradient in SW28 tubes by        carefully layering 5 ml of 40%,    -   5 ml of 30%, 6 ml of 20%, 8.5 ml of 10%, and 8.5 of 5% sucrose        solutions in buffer A from bottom to top.    -   3. CCVs (5-10 ml) is laid on top of the gradient and centrifuged        at 100,000 g (25,000 rpm) in a SW28 rotor for 1 hr at 4° C.    -   4. Collect twenty-six 1.5 ml factions from the top.    -   5. Small aliquots from every other faction are analyzed for CCVs        using 10% SDS-PAGE. [Fractions comprising the CCVs (typically        fractions 12-21 as numbered from the top of the gradient) are        combined, diluted with 3 volumes of buffer A, and centrifuge at        112,000 g (31,100 rpm) in a ti 45 Beckman rotor for 1h at 4° C.        or at 33,000 rpm in a Beckman 70.1Ti rotor for 1h at 4° C. Add        1:100 PMSF]    -   6. Resuspend the pellet in ice-cold buffer A, do a protein assay        to yield an approximate concentration. Usually add 1 to 2 ml of        buffer A.    -   7. Aliquot the homogenate in aliquots of 200 ul and store at        −80° C. Take an aliquot of 10 ul each for EM and SDS-gel PAGE.        Part III. Isolation of Triskelia and APs from CCVs Using Keen's        Method.    -   1. Dialyze CCVs against 0.01M Tris buffer, Ph 8.5, 3 mM azide        for 5 hours.    -   2. Centrifuge at 240,000 g (51,200 rpm) for 20 min at 4° C.        Because you are using low amount of sample; (IF we have less        than 2 mL, Do not use the lid or close the centrifuge tubes of        the 70.1 Ti rotor.) The soluble coat proteins comprising        triskelial and APs are separated from the residual Clathrin-coat        vesicle membranes.    -   3. Collect the soluble fraction and do protein assay.    -   4. Take an aliquot of 10 ul for EM and 50 ul for SDS-gel PAGE.        Part IV. Separation by FPLC of AP-1 from AP-2 with        Hydroxyapatite Column

Solutions:

Stocks: 1M NaH₂PO₄; pH 7.1 (30 g/250 ml)  5M NaCl   10% NaN₃ Low PO₄buffer (500 ml): 10 mM NaH₂PO₄; pH 7.1  (5 ml of stock) 100 mM NaCl  (10ml of stock) 0.02% NaN₃  (1 ml of stock) 0.1% beta-Mercaptoethanol (0.5ml) (RT) High PO₄ buffer (200 ml): 500 mM NaH₂PO₄; pH 7.1 (100 ml ofstock) 100 mM NaCl  (4 ml of stock) 0.02% NaN₃  (0.4 ml of stock)  0.1%beta-Mercaptoethanol (0.2 ml)

-   -   Both buffers need to be filtered and degassed prior to use.

AP buffer: 100 mM MES, pH 7.0   39 g/2 l 150 mM NaCl 17.5 g/2 l 1 mMEDTA 4 ml of 500 mM solution/2 l 0.02% NaN₃ 4 ml of 10% solution/2 l 0.5mM DTT → add just before use (4° C.)

-   -   Hydroxyapatite Column:        5 ml Econo-Pac CHT-II from BioRad; the column is stored at 4° C.        in low PO₄ buffer

Procedure:

-   -   Connect the hydroxyapatite column to the FPLC system via the        BioRad adaptors. Put a 0.2μ syringe filter at the inlet of the        column.    -   Use the following FPLC settings:        -   Sensitivity: 1        -   Flow: 1 ml/min        -   Chart Recorder speed: 0.5 cm/min        -   Make sure the fraction collector is set at “ml” and a volume            of “1”    -   Pump A is used for the low PO₄ buffer; Pump B for the high PO4        buffer. Wash the pumps with Valve 1 in position “3”.    -   Once the FPLC system is set up, start washing the column with 20        ml of high PO₄ buffer (=20 min). Be sure to switch on UV-Lamp.    -   This is followed by equilibration of the column with low PO₄        buffer; i.e. until the baseline is stable. The backpressure of        the system should be approx. 0.1 MPa and must not exceed 0.35        Mpa.    -   During the equilibration phase (Valve 1 in position “1”=“Load”),        the 50 ml superloop is loaded with the AP sample (Pump C; 5        ml/min).    -   With the column equilibrated and the superloop loaded, switch        Valve 1 into position “2”=“Inject”. The APs are injected over        the column at a flow rate of 1 ml/min.    -   After the injection is completed, continue running low PO₄        buffer over the column until the baseline is stable. Don't        forget to prepare 1.5 ml tubes for the fraction collector.    -   AP-1 and AP-2 are then eluted from the column using Method 6:

 0.0 CONC % B 0.0  0.0 VALVE.POS 1.1  0.0 CM/ML 0.50  0.0 PORT.SET 6.140.0 CONC % B 0.0 40.0 ML/MIN 1.00 50.0 CONC % B 100

The elution profiles for AP-1 and AP-2 tend to vary considerably fromone purification to another; AP-1 is eluted first.

-   -   AP-1 tends to be eluted from the column in three to four 1 ml        fractions, usually starting at around #13. AP-2 is usually        eluted in up to 15 fractions, starting at around #25. The        fractions comprising the APs need to be verified by SDS-PAGE        (two gels of 10% or 12%)    -   Wash column with low PO₄ buffer; store at 4° C.    -   Pooled AP-1 fractions and pooled AP-2 fractions are dialyzed        against 1 liter of AP buffer overnight, and for a few more hours        after exchanging the buffer (4° C.). The samples are then stored        at 4° C.    -   Typically, the concentration for Clathrin (peak fractions) is        approx. 0.5 mg/ml, for AP-1 and AP-2 between 0.3-0.5 mg/ml.

According to one illustrative embodiment, but is not limited to,recombinant Clathrin formation may be achieved in the following exemplarmanner. Stoichiometric quantities of adaptor elements 208 a comprisingAP-1 and AP-2 are required for Clathrin self-assembly at physiologicalpH. However, in vitro Clathrin self-assembly occurs spontaneously belowabout pH 6.5. Recombinant terminal and distal domain fragments areproduced and combined with recombinant-produced hub fragments inassembly buffer as described below in order to induce formation of oneor more Clathrin elements, such as those comprising elements 206 a, foruse in the invention.

In one illustrative technique, bovine Clathrin heavy chain cDNA encodingheavy chain amino acids 1-1074 (SEQ ID NO: 1) is cloned into the pET23dvector (Novagen) between the NcoI (234) and XhoI (158) sites. Expressionof the cloned sequence results in a terminal and distal domain fragmentshaving a C-terminal polyhistidine tag. Hub fragments corresponding toamino acids 1074-1675 (SEQ ID NO: 2) are cloned into vector pET15b(Novagen) between the BamHI (319) and XhoI (324) sites. Expression ofthe hub fragments produces the proximal leg domain and centraltrimerization domain of the Clathrin hub with an N-terminalpolyhistidine tag. Vectors comprising the heavy chain and hub domainsare expressed in E. coli by induction with 0.8 mMisopropyl-B-D-thiogalactopyranoside for 3 hours at 30 degrees Celsius.Expressed proteins are isolated, recombinant, and or synthetic frombacterial lysate in binding buffer (50 mM Tris-HCl (pH7.9), 0.5M NaCl, 5mM imidazole) in a nickel affinity resin using the polyhistidine tag.Proteins are eluted with 206 a mM EDTA and dialyzed against 50 mMTris-HCl (pH7.9). Hub fragments are further isolated, recombinant, andor synthetic using size exclusion chromatography on a Superose 6 column(Pharmacia).

In another exemplar technique, Clathrin assembly reactions are performedusing expressed heavy chain and hub fragments by overnight dialysis at 4degrees Celsius in assembly buffer (100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.7, 0.5 mM MgCl2, 1 mM EGTA, 1 MM Tris(2-carboxyethyl)-phosphine hydrochloride, 3 mM CaCl2. Assembly reactionsare centrifuged for 5 minutes at 12,000 rpm. The supernatant is thencentrifuged for 45 minutes at 45,000 rpm (100,000×g). The pellets areresuspended in assembly buffer, and protein composition is determined onSDS-PAGE. The efficiency of element 206 a formation can be determined byelectron microscopy by diluting assembly reactions 1:5 in 10 mM TrispH7.9, and placing aliquots on a glow-discharged carbon-coated grid,using 1% uranyl acetate as the stain.

According to another illustrative embodiment, but is not limited to,recombinant Clathrin formation may be achieved in the following exemplarmanner, as described by Rapoport, et al. (MBC 2008): A cDNA encoding ratClathrin heavy chain (Kirchhausen et al., 1987a) is used as a templateto generate full-length (1675 HC), nested C-terminal truncations (1661HC, 1643 HC, 1637 HC, 1630 HC, and 1596 HC), internal deletions (1675PIVYGQ HC, 1643 PIVYGQ HC, and 1675 QLMLTA HC), and mutations(1643LML-AAA HC) of the heavy chain; each is then subcloned into theinsect cell expression vector pFastBac1 (Invitrogen, Carlsbad, Calif.).A cDNA encoding rat liver Clathrin light chain LCa (Kirchhausen et al.,1987b) is used as the template to subclone the region encoding the fulllight chain (residues 1-256) into the insect cell expression vectorpFastBacHTb. The final construct (rLCa1i) comprises at its N terminus a6×-His-tag followed by a linker of 20 residues. Baculoviruses suitablefor infection and expression are generated with the Bac-to-Bac system(BD Biosciences, San Jose, Calif.). Virus stocks are obtained after fourrounds of amplification, and they are kept in the dark at 4° C. The openreading frame of rat brain Clathrin light chain LCa1 is also used as atemplate to subclone it into the bacterial expression vector pET28b(Novagen, Madison, Wis.) between the NcoI and EcoRI restriction sites soas to generate a native, nontagged light chain. All constructs areverified by DNA sequencing. Clathrin heavy chains together with lightchain are expressed in Hi5 insect cells (1 L, 1-1.5 206 a cells/ml)grown for 2-3 d in spinner flasks at 27° C. in Excell 420 medium aftercoinfection with the appropriate viruses. Alternatively, Clathrin heavychain only is expressed in a similar way. The cells are centrifuged at1000 rpm for 10 min at room temperature by using an H6000A rotor(Sorvall, Newton, Conn.), and the pellets are resuspended in 20 ml lysisbuffer (50 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 3 mMmercaptoethanol, and half of a tablet of Complete Protease InhibitorCocktail [Roche Applied Science, Indianapolis, Ind.]). The resuspendedpellets are sonicated for 1 min on ice (Flat tip at 20% power,Ultrasonic processor XL; Heat Systems, Farmingdale, N.Y.), cell debrisis removed by centrifugation at 90,000 rpm for 20 min at 4° C. by usinga TLA 100.4 rotor (Beckman Coulter, Fullerton, Calif.), and thesupernatant (20 ml) is dialyzed at 4° C. for 12 h against 2×2 l of cagebuffer (20 mM [2-(N-morpholino) ethanesulfonic acid] MES, pH 6.2, 2mMCaCl2, 0.02% NaN3, and 0.5m Mdithiothreitol [DTT]). The sample is thencentrifuged at 4° C., first at low speed (1000 rpm for 10 min) to removelarge aggregates and then at high speed (54,000 rpm for 1 h) by using aTi rotor (Beckman Coulter). The pellet, primarily comprising Clathrin(presumably assembled as cages) is resuspended in 6 ml of 100 mM MES, pH6.5, 3 mM EDTA, 0.5 mM MgCl2, 0.02% NaN3, 0.5 mM DTT, and 0.5 mMphenylmethylsulfonyl fluoride) followed by addition of 3 ml of 2.4MTris,pH 7.4, 1 mM DTT, and incubation for 20 min at room temperature, acondition used to dissociate native Clathrin assemblies. The sample iscentrifuged at 90,000 rpm for 20 min at 4° C. by using a TLA 100.4rotor, and most of the Clathrin is recovered in the supernatant. Theresulting sample is subjected to gel filtration chromatography (90cm×0=3 cm column comprising Sephacryl-S 500 [GE Healthcare, LittleChalfont, Buckinghamshire, United Kingdom] in 0.5 M Tris, pH 7.4, 0.04%NaN3, and 0.5 mM DTT) at room temperature and with a flow of 2 ml/min.Fractions of 5.5 ml comprising the Clathrin peak (100 ml) are pooled andthen subjected to adsorption chromatography (5 ml, hydroxyapatite,Econo-Pac CHT-II; Bio-Rad, Hercules, Calif.); the column ispre-equilibrated with low phosphate buffer (10 mM NaH2PO4, pH 7.1, 100mM NaCl, 0.02% NaN3, and 0.5 mM DTT) and eluted with a linear gradientfrom low to high phosphate concentration (500 mM NaH2PO4, pH 7.1, 100 mMNaCl, 0.02% NaN3, and 0.5 mM DTT) at room temperature with a flow of 1ml/min. Fractions (1 ml) are collected into microcentrifuge tubescomprising 2 l of 0.5 M EDTA. Typical Clathrin yields are in the rangeof 3-40 mg per 1 l of cell culture. Western blot analysis is used toconfirm the expression of Clathrin heavy and light chains. The ratClathrin light chain rLCa1b is expressed in Escherichia coli strainBL21(DE3). The bacteria are grown in Luria-Bertani (LB) mediumcomprising 30 mg/l kanamycin at 37° C. with shaking (250 rpm) to anoptical density of 0.5. Expression is induced by addition ofisopropyl-d-thiogalactoside (IPTG) (final concentration, 0.6 mM). After3 h, the cell are harvested by centrifugation at 5000 rpm for 10 min at4° C. by using an H6000A rotor (Sorvall) and resuspended in ice-coldlysis buffer (20 mM Bis-Tris adjusted to pH 6.0 at room temperature, 0.5mM dithiothreitol, 1 mM EDTA, and Complete Protease Inhibitor Cocktail)by using 20 ml of lysis buffer per 3.5 g of wet cell weight. Thesuspension is placed into a glass vessel, and the vessel is immersed inboiling water for 4 min and then chilled on ice. The boiled suspensionis centrifuged at 54,000 rpm for 30 min at 4° C. by using a 60Ti rotor(Beckman Coulter) to remove the precipitated material. rLCa1b ispurified from the filtered supernatant (0.2-msyringe filter) by anionexchange chromatography at 4° C. on a HiTrap MonoQ column equilibratedwith buffer A (20 mM Bis-Tris, adjusted to pH 6.0 at room temperature,and 0.5 mM dithiothreitol) and eluted using a linear gradient from 0 to32% buffer B (20mMBis-Tris, adjusted to pH 6.0 at room temperature, 0.5mM dithiothreitol, and 1 M NaCl). For the in vitro reconstitution ofClathrin, recombinant heavy chain (expressed in insect cells withoutlight chain) is mixed with excess rLCa1b (expressed in bacteria) byusing a weight ratio of 3:1 (equivalent to a molar ratio HC:LC of 1:2.4)just before cage or coat assembly for 40 min at room temperature.

Part V. Clathrin Coat Formation

Reagents

1. Coat Formation Buffer

80 mM Mes hydrate pH 6.5 31.23 g/2 L 20 mM NaCl  2.34 g/2 L 2 mM EDTA  8mL of 500 mM stock solution/2 L 0.4 mM DTT 1.6 mL of 500 mM stocksolution/2 L

2. Clathrin 3. AP-2 Procedure

(1) Place a solution of clathrin and AP-2 into a dialysis chamber

-   -   clathrin: AP-2=3:1 to 4:1 (w/w)        (2) Dialyze over night against coat formation buffer; replace        buffer and dialyze for an additional 3-4 h.        (3) Transfer to a centrifuge tube, centrifuge to remove larger        aggregates    -   rotor: TLA-100.4, 12000 rpm, 4° C., 10 min        (4) Transfer supernatant to fresh centrifuge tube, centrifuge to        collect coats    -   rotor: TLA-100.4, 65000 rpm, 4° C., 12 min        (5) Immediately withdraw supernatant with a 1 mL pipette.        (6) Wash carefully with buffer around the pellet.        (7) Resuspend the pellet by adding buffer, allowing to stand at        room temperature for 10-15 min, then slowly wash buffer over the        pellet to resuspend using a micro-pipettor (avoid foaming)    -   volume: 120-150 μL for a pellet of ˜3 mm diameter

Part VI. Clathrin Cage Formation Reagents 1. Cage Formation Buffer:

20 mM Mes, pH 6.2 (3.9 g/l) (7.8 g/2l)

2 mM CaCl2 (2 ml of 1M/1) (4 ml of 1M/2l)

0.02% NaN3 (2 ml of 10%/1) (4 ml of 10%/2l)

0.5 mM DTT (1 ml of 500 mM/1) (2 ml of 500 mM/2l)

2. Clathrin Procedure

(1) Place a solution of Clathrin (0.5-1 mg/mL) into a dialysis chamber(2) Dialyze over night against cage formation buffer; replace buffer anddialyze for an additional 3-4 h.(3) Transfer to a centrifuge tube, centrifuge to remove largeraggregates

-   -   rotor: TLA-100.4, 12000 rpm, 4° C., 10 min        (4) Transfer supernatant to fresh centrifuge tube, centrifuge to        collect coats    -   rotor: TLA-100.4, 65000 rpm, 4° C., 12 min        (5) Immediately withdraw supernatant with a 1 mL pipette.        (6) Wash carefully with buffer around the pellet.        (7) Resuspend the pellet by adding buffer, allowing to stand at        room temperature for 10-15 min, then slowly wash buffer over the        pellet to resuspend using a micropipettor        (avoid foaming)

Production of Recombinant Auxilin

A protein chimera of glutathione transferase (GST) with bovine auxilin(spanning residues 547-910) is generated by fusion in the vectorpGEX4T-1 and then used for expression in E. coli BL21 (Fotin et al.,2004a). The bacteria are grown in LB medium supplemented with ampicillinto an OD600 0.5-0.6 at 37° C. Protein expression is induced by additionof 1 mM IPTG (final concentration) and the cells grown for another 4 hat 25° C. The cells (from 1 l of culture) are centrifuged at 5000 rpmfor 15 min at 4° C., and the pellet is kept frozen overnight. The pelletis resuspended in 25 ml of pGEX lysis buffer (20 mM HEPES, pH 7.6, 100mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, and half a tablet ofComplete Protease Inhibitor Cocktail) and sonicated on ice using threeconsecutive sonication cycles of 60, 30, and 30 s (standard microtip,20% power). The sample is centrifuged at 45,000 rpm for 1 h at 4° C. byusing a 60Ti rotor, and the supernatant mixed with 0.5 ml of a 50%(vol/vol) slurry of glutathione-Sepharose 4 beads (GE Healthcare). After2 h of end-over-end rotation at 4° C., the beads are poured into apropylene Econo-Column (Bio-Rad), washed with 15 ml of pGEX lysisbuffer, and then washed with 15 ml of 25 mM HEPES, pH 7.0, 100 mM NaCl,and 0.1 mM EGTA. Elution of GST-auxilin (in 2 ml) is achieved bysupplementing the solution with 50 mM glutathione, adjusted to pH 8.These steps are carried out at 4° C. Release of the GST portion isachieved by incubation of 1 mg of GST-auxilin with 1 U of thrombin atroom temperature for 6 h. Proteolysis is ended by addition of 1 mg ofPefabloc SC (Roche Applied Science). The 40-Da auxilin fragment isfurther purified using a Mono S column (Pharmacia, Peapack, N.J.). Thesample is first dialyzed overnight against MES buffer A (50 mM MES, pH6.7, 1 mM EDTA, and 3 mM-mercaptoethanol), and then it is loaded ontothe column (pre-equilibrated with MES buffer A) and eluted with a lineargradient of buffer A and with MES buffer B (50 mM MES, pH 6.7, 500 mMNaCl, 1 mM EDTA, and 3 mM-mercaptoethanol) at a flow of 1 ml/min. Theauxilin sample is stored at 80° C. with 20% glycerol (finalconcentration).

Production of Recombinant Hsc70

N-terminal 6×-His-tagged bovine Hsc70 (full length) cloned into thepET21avector is expressed in E. coli BL21. The bacteria are grown at 37°C. in LB supplemented with 0.1 mg/ml ampicillin to an OD600 of 0.5,transferred to 28° C., and induced with 0.1 mM IPTG for 5 h. The cellsare centrifuged at 5000 rpm for 15 min at 4° C., and the pellets from 11culture resuspended in 25 ml 50 mM Tris, pH 8.0, 300 mM NaCl, 1 mM ATP,2 mM MgCl2, 10 mM-mercaptoethanol, and half a tablet of CompleteProtease Inhibitor Cocktail without EDTA. The supernatant obtained aftersonication and centrifugation (as with auxilin) is mixed with 1 ml of50% (vol/vol) slurry of nickelnitrilotriacetic acid-agarose beads(QIAGEN, Valencia, Calif.) for 4 h by endover-end rotation at 4° C. Thebeads are placed into an Econo Pac column and then washed with 30 ml of50 mM Tris, pH 8.0, 300 mM NaCl, 10 mM-mercaptoethanol, 10 mM imidazole,1 mM ATP, and 1 mM MgCl2). Hsc70 is then eluted at 4° C. with 5-6 ml ofthe same solution supplemented with 200mMimidazole. Fractions of 1 mlare collected into microcentrifuge tubes comprising 40 l of 0.1 M EGTA.The samples comprising 20% glycerol (final concentration) are stored at80° C.

According to another illustrative embodiment, Clathrin and or CoatomerI/II proteins are extracted and prepared from Clathrin and or CoatomerI/II coated vesicles obtained from non-rat, non-bovine organic tissue,including from human tissue, in whole or in part. In another embodiment,Clathrin and or Coatomer I/II coated proteins are extracted and preparedfrom Clathrin and or Coatomer I/II coated vesicles obtained bydonor/recipient tissue matching using established techniques. In anotherembodiment, Clathrin and or Coatomer I/II proteins are prepared, inwhole or in part, by using stem cells, cloning and or other geneticmanipulation techniques known in the prior art to produce geneticallymatched tissue for a donor recipient.

According to one illustrative embodiment, the coat protein I (COPI)assembly process is carried out by preparing Coatomer subunits fromcytosolic preparations, including methods, but are not limited to, asessentially described in Spang, et al., Proc. Natl. Acad. Sci. USA. 1998Sep. 15; 95 (19): 11199-11204. Coatomer, a nanoscale element comprisedof seven distinct subunits (alpha, beta, beta′, gamma, delta, epsilonand zeta subunits, respectively) and ADP-ribosylation factor (ARF, anN-myristylated small GTP-binding protein) are the only cytoplasmicproteins needed.

In another illustrative embodiment, the coat protein I (COPI) assemblyprocess is carried out by preparing Coatomer subunits from cytosolicpreparations, including methods, but are not limited to, as essentiallydescribed in Sheff, et al, The Journal Of Biological Chemistry, Vol.271, No. 12, Issue Of March 22, Pp. 7230-7236, 1996 “Purification of RatLiver Coatomer (COPI”)—Purification of rat liver Coatomer isaccomplished through a substantial modification of the method of Watersand Rothman (13). Unless otherwise noted, all operations are performedat 4° C. Approximately 250 g of fresh liver from 10-15 adultSprague-Dawley rats (Harlan Sprague-Dawley) are homogenized in 2 volumesof buffer (25 mM Tris, pH 7.5, 320 mM sucrose, 500 mM KCl, 2 mM EDTA, 1mM dithiothreitol) comprising protease inhibitors (2 mg/ml pepstatin A,antipain, and leupeptin; 1 mM phenylmethylsulfonyl fluoride) using apolytron homogenizer with 1.5-cm cutter assembly at maximum speed forthree 1-min bursts on ice with 1-min rests. The lysate is cleared bysequential centrifugation at 9000 3 g for 15 min followed bycentrifugation of the supernatant at 100,000 3 g for 1 h. This material(S100) is stored at 270° C. for up to 4 months. For a typicalpurification, 150 ml of S100 is diluted 6-fold with cytosol buffer (25mM Tris, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA plus protease inhibitorsas above). Protein concentration is 5 mg/ml. Ammonium sulfate is addedto 25% of saturation and stirred for 15 min on ice, and then precipitateis removed by centrifugation, and the supernatant is brought to ammoniumsulfate at 45% of saturation with stirring on ice. The precipitate iscollected by centrifugation and redissolved in 150 ml of cytosol buffer.An additional 120 ml of cytosol buffer is added and then 30 ml of 60%(w/v) polyethylene glycol 3350 in distilled H₂O with gentle stirring.The mixture is incubated at 4° C. for 30 min, and the precipitate iscollected by centrifugation at 10,000 3 g for 15 min. The precipitate isresuspended in 20 ml of G buffer (10 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mMCaCl2), the insoluble material is removed by centrifugation, and thesupernatant is passed over a 20-ml column comprising 250 mg of DNase-I(Sigma) coupled to agarose (Affi-Gel-10, Bio-Rad, prepared according tothe manufacturer's directions) to remove contaminating actin and actinbinding proteins. Eluent is desalted into cytosol buffer using 10DGdesalting columns (Bio-Rad) and applied to a 50-ml DEAE cellulose column(DE52, Whatman) equilibrated in cytosol buffer. COPI is eluted with a100-400 mM KCl gradient over 200 ml, with the elution of COPI followedby spot blot on nitrocellulose using EAGE antibody. In a final step,peak COPI fractions are pooled, diluted 1:1 with cytosol buffer, andapplied to a 1-ml Mono-Q column (Pharmacia) equilibrated in cytosolbuffer and mounted on a fast protein liquid chromatography apparatus(Pharmacia). The column is swished with 300 mM NaCl and then eluted witha 350-400 mM NaCl gradient over 20 ml. COPI, as assayed by the presenceof b-COP on a spot blot using EAGE antibody, eluted as a single peak.The presence and purity of COPI is confirmed by SDS-PAGE. An alternativefinal step is employed in preparing samples for two-dimensionaldimensional gels. Here, DEAE eluent is concentrated in a Centricon-30microconcentration (Amicon) to 400 ml and applied to a 24-ml Superose-6(Pharmacia) column equilibrated in cytosol buffer with 50 mM KCl. Aswith Mono-Q, COPI eluted in a single peak. This final step produces asomewhat lower yield and comprises some contaminants between 30 and 100KD by SDS-PAGE. For copurification of labeled CHO cytosol and rat liverCOPI, all quantities are divided by 3, 1 ml of labeled cytosol is addedto 50 ml of rat liver S100, and the Mono-Q column is used as the finalstep.

The increasing interest in the targeting of foreign moieties at sites inthe body where their activity is required is addressed by the inventionin one more embodiments. It is important that agents, like drugs,particularly those having undesirable side effects, are delivered to thesite where they are supposed to act. Many molecular species require thatthey be delivered in a site specific manner, often to particular cells,for example, polynucleotides (anti-sense or ribozymes), metabolicco-factors or imaging agents. One such system has been described by Wuet al., J. Biol. Chem., 263, 14621-14624 and WO-A-9206180, in which anucleic acid useful for gene therapy is conjugated with polylysinelinked to galactose which is recognized by the asialoglycoprotein cargoattachment elements on the surface of cells to be targeted. However,there are many occasions, such as in the delivery of a cytotoxic drug,when it would not be satisfactory to use a delivery system in which thetargeting and or masking moiety and or vector to be delivered is soexposed. This need is addressed by various delivery system embodimentsof the invention that possess the flexibility to target a wide range ofbiologically active foreign moieties.

In one embodiment, the invention includes one or more elements havingone or more suitable sites for subsequent attachment of a targeting andor masking moiety and or vector, and one or more elements having one ormore surfaces and or protein coats to which one or more targeting and ormasking moieties and or vectors have already been attached.

In one embodiment, one or more masking moieties are attached to thesurface of one or more invention elements. These masking moietiesprevent the recognition by a specific cell surface and instead allowsfor intravenous administration applications. For example, the surfacemasking characteristics may be provided by poly (ethylene glycol) (PEG)by using various PEG-PLA and PLGA mixtures. PEG conjugation masks theprotein's surface, reduces its renal filtration, prevents the approachof antibodies or antigen processing cells and reduces its degradation byproteolytic enzymes. In one embodiment, PEGylated elements significantlyimprove element stability and prevent leakage of agents from elements.Studies have shown that protein-based nanoparticles and liposomeswithout PEGs have a short circulation time due to rapid uptake bymacrophages of the reticulo-endothelial system (RES), primarily in theliver and spleen. Finally, PEG conveys to molecules its physico-chemicalproperties and therefore modifies biodistribution and solubility ofpeptide and non-peptide nanoparticles. Thus, recent studies have usedmostly nanoparticles with PEGs. The PEG coating is highly hydrated andthis layer protects against interactions with molecular and biologicalcomponents in the blood stream, as well as nonspecific binding totissue. In one embodiment, one or more elements, in one or moreconfigurations, are internally and or externally attached, coated, andtreated, in whole or in part by using steric stabilizers including, butnot limited to, steric stabilizers selected among dipalmitoylphosphatidyl ethanolamine-PEG, PEG-stearate, the esters of the fattyacids from the myristic acid to the docosanoic acid with methyl etherPEG, the diacylphosphatidyl ethanolamines esterified with methyl etherPEG and the polylactates and the polyglycolactates esterified withmethyl ether PEG. In one embodiment, one or more elements are notrequired to be PEGylated to efficaciously operate.

In another embodiment, one or more elements, and in one or moreconfigurations are internally and or externally coated or treated inwhole or in part with surfactants, including, but not limited to,surfactant agents selected among soy-bean phosphatidylcholine, dioleylphosphatidylcholine, dipalmitoyl phosphatidylcholine, hydrogenatedsoy-bean phosphatidylcholine, phosphatidylethanolamine andphosphatidylserine), and or with cosurfactants, including, but notlimited to cosurfactant agents selected among ethanol, propanol,isopropanol, butanol, sodium taurocholate, sodium glycocholate,propylene glycol, butyric acid and benzoic acid.

In one or more embodiments, ligands can be of one or more efficacioustypes, such as drugs, and may be bioengineered, and or compriseisolated, recombinant, synthetic, and or cloned elements.

In one embodiment, one or more types of ligands may be functionalizedand or attached in one or more ways to one or more elements.

In one embodiment, ligands are natural ligands of one or more types. Inanother embodiment, one or more types of natural ligands are modifiedand or functionalized. In another embodiment, invention element ligandsand natural element ligands are combined to comprise one or more typesof hybrid ligand elements.

In another embodiment, the course of a natural ligand and or inventionligand element during cellular signaling, trafficking, downregulation,upregulation, endocytosis, exocytosis, and other cellular entry or exit,cellular inter- and or intra-actions, and the like, may be efficaciouslycontrolled, regulated, and or modified by one or more elements to yieldone or more diagnosis, cure, mitigation, treatment, prevention ofdisease, or other types of efficacious effects, and the like.

Examples of some natural ligands, but not limited to, that may besubject to efficacious control, modification, and or regulation in oneor more invention embodiments are listed below:

-   -   Toxins and lectins, e.g.,    -   Diptheria Toxin    -   Pseudomonas toxin    -   Cholera toxin    -   Ricin    -   Concanavalin A    -   Viruses, e.g.,    -   Rous sarcoma virus    -   Semliki forest virus    -   Vesicular stomatitis virus    -   Adenovirus    -   Influenza    -   West Nile    -   Serum transport proteins and antibodies, e.g.,    -   Transferrin    -   Low density lipoprotein    -   Transcobalamin    -   Yolk proteins    -   IgE    -   Polymeric Ig    -   Maternal Ig    -   IgG, via Fc receptors    -   Hormones and Growth Factors, e.g.,    -   Insulin    -   Epidermal Growth Factor    -   Growth Hormone    -   Thyroid stimulating hormone    -   Nerve Growth Factor    -   Calcitonin    -   Glucagon    -   Prolactin    -   Luteinizing Hormone    -   Thyroid hormone    -   Platelet Derived Growth Factor    -   Interferon    -   Catecholamines    -   LDL    -   Neurotransmitters    -   Substance P

A Neurotransmitter Known to Stimulate Pain Receptors

In one or more embodiments, one or more elements are conjugated (bonded)with one or more other elements (e.g., ligands), agents, materials, andor substances of one or more types, including those developed by 3^(rd)parties, which may be used singly or mixed together in one or moreconfigurations for medical and biological research, diagnosis, therapy,or prosthetic purposes. One or more biomedical elements such as ligandsand other types of biomedical functionalization elements may be directlyand or indirectly attached, bonded, fastened, cross-linked, and oraffixed to and or incorporated into one or more invention elements, aswell as one or more non-invention and or natural elements. In oneembodiment, attachment is achieved via molecular tethers. In anotherembodiment, no molecular tether is involved. In one configuration, afree radical molecule may be attached directly to one or more inventionelements. In another embodiment, one or more elements may be bonded,fastened, and or affixed to one or more elements by being included in amodified protein sequence of one or more elements or bonded elements; byusing a spacer; by covalent bonding; by site directed mutagenesis; bygenetically engineered mutation and or modification; by peptides; byproteins; by DNA; by antibodies; by monoclonal antibodies; byrecombinant elements; and via other bioengineering techniques andmethods known in the art.

According to one embodiment, the protein amino acid sequence of one ormore elements are modified to provide a site suitable for attachmentthereto of an in vivo or in vitro targeting and or masking moiety. Inone illustrative embodiment, one or more target-specific ligands and ortargeting moieties are directly attached to one or more elements via oneor more amino acid groups, and or attached via one or more shortmolecular tethers.

In another embodiment, one or more functionalization elements, of one ormore types, comprise highly specific targeting agents, such as, but notlimited to, antibodies, peptides or small molecules, large molecules,and other functional ligands, such as fluorophores and permeationenhancers, and the so functionalized nanoparticles may target receptors,transporter, enzymes and or intracellular processes in vivo with highaffinity and specificity.

In one illustrative embodiment, one or more elements such as diagnostic,therapeutic, prosthetic, and or assay agents, but not limited to, aredelivered to a target in vivo or in vitro using a variety of guidancetechniques, including for example, optical (photonic), acoustic,electric, biological, chemical, mechanical reactions and forces, but notlimited to, and one or more elements may be delivered singly and or inone or more configurations to one or more targets.

In another illustrative embodiment, one or more elements comprise one ormore diagnostic agents like imaging contrast or radioactive agents toperform site designation, site specificity, and site retention fortargeted in vivo delivery of therapeutics; the latter may also comprisepart of the same diagnostic payload.

In one illustrative embodiment, the invention enables targeted agentdelivery systems that retain their structural integrity and that mayalso loiter for a calculated period of time at the targeted area ofconcern after delivery of agent payload.

In one illustrative embodiment, one or more elements comprise moleculesarranged in specific patterns. The pattern of elements precisely mirrorsor mimics a spatial or physical pattern a target cell in a human oranimal body expects to see and will recognize, and one or more elementsare accepted by the target cell, which can be a cancer cell or HIVinfected cell, for example.

In one embodiment, gold metal nanoparticle probes with sensor ligandsand using electrical charges are bonded to one or more elements, and orattached to ligands, targeting moieties, and or vectors. The goldparticles carry short strands of artificial DNA (oligonucleotides)tailored to match known segments of biological DNA that are implicatedin, or linked to, disease.

Target-specific ligand binding and any subsequent changes within or toone or more elements may be a result of either covalent or non-covalentinteractions—the latter including hydrogen bonding, ionic interactions,Van der Waals interactions, and hydrophobic bonds—depending on theapplication, system design, receptor design, cargo type and or theinteraction/application environment.

In another illustrative embodiment, one or more elements, ligands,targeting moieties, vectors, and the like utilize the method ofchirality.

In another illustrative embodiment, reactions and forces arise from oneor more ligands and or targeting moieties binding to targets, includingcovalent and non-covalent interactions, which ligands are tethered andor directly attached to one or more invention elements. Ligand bindingto one or more specific targets may produce one or more conformationalchanges sufficient to deform and or rupture one or one or more elementsin whole or in part, thereby causing one or more elements to bereleased. The targeting moieties can be selected by one of ordinaryskill in the art keeping in mind the specific cell surface to betargeted. For example, if one wishes to target the asialoglycoproteinreceptor on the hepatocytes in the liver, an appropriate targetingmoiety would be clustered trigalactosamine. Once a specific targetingmoiety has been selected for a particular cell to target, the differenttargeting moieties can be attached either by covalent linkage directlyonto the surface of one or more invention elements, or by indirectlinkage via, for example, a biotin-avidin bridge. In another embodiment,depolymerization (e.g., by cytosolic Hsc 70) of the Clathrin and orCoatomer element exposes one or more transmembrane proteins (V-SNARE)that direct one or more elements to their destinations by binding to aspecific T-SNARE protein on the target organelle. The fusion proteinSNAP25 causes the one or more elements to fuse with the target membrane

In one embodiment, avidin is attached covalently to the surface of oneor more elements and a biotinylated ligand attaches non-covalently tothe avidin. In another embodiment, biotin is covalently attached to thesurface of one or more invention elements, and then avidin is used as abridge between the biotinylated polymer and the biotinylated ligand.Targeting agents may also include one or more biocompounds, or portionsthereof, that interact specifically with individual cells, small groupsof cells, or large categories of cells. Examples of useful targetingagents include, but are not limited to, low-density lipoproteins(LDS's), transferrin, asiaglycoproteins, gp120 envelope protein of thehuman immunodeficiency virus (HIV), and diphtheria toxin, antibodies,and carbohydrates. A variety of agents that direct compositions toparticular cells are known in the prior art (see, for example, Cotten etal., Methods Enzym, 1993, 217, 618).

In another illustrative embodiment, one or more classical structuralactivity relationships (SARs) based drug discovery approaches arecombined with one or more other techniques to form a specific case oftargeted drug delivery, for example, but not limited to, one or morestructural metabolism relationships (SMRs) that in combination with SARsare sometimes termed as retrometabolic drug design approaches. Theseactive drugs are designed to undergo singular metabolic deactivationafter they achieve their therapeutic roles, and may produce specificaction at the site of application without affecting the rest of thebody.

In another illustrative embodiment, one or more elements comprise one ormore agent functionalities and or methods that produce targeting bychanging molecular properties of an overall target molecule, as a resultof enzymatic conversion, but also, for example, may involve one or morepharmacophores. These elements, sometimes referred to as the targetor(Tor) moiety, are converted by site-specific enzymes to activefunctions. In addition to the Tor moiety, one or more other functionsmay be introduced into elements for in vivo use, which can be named as“protector functions” that serve as lipophilicity modifiers orprotectors of certain functional groups in therapeutic agent molecules.

In other illustrative embodiments, one or more other types of targetingdelivery systems and methods n be used, for example, but not limited to,in whole or in part in one or more configurations: surfactants(surface-active substances) and or cosurfactants; enzymaticphysical-chemical-based targeting; site-specific enzyme-activatedtargeting; vectors, such as ligand-based, non-viral-based, andProtein/DNA polyplex vector targeting; receptor-based chemicaltargeting; organic and or inorganic synthetic elements; transmembraneproteins (V-SNARE); peptides, including peptides that cross cellmembranes and home specifically to certain diseases; nanostructureddendrimers and hyperbranched polymers; molecular Trojan horses;adenovirus, herpes simplex virus, adeno-associated virus or other virusvectors for targeted delivery that do not cause toxicity; antibodies,including monoclonal antibodies; nanoparticles, including polymernanoparticles like polymer, polybutylcyanoacrylate, and ethyl alcoholnanoparticles; immunotoxins; hormonal therapy; tissue-specific geneexpression; gene therapy; pegylated immunoliposomes; anti-sense therapy;biological elements and or agents, including biological elements andagents conjugated with other agents, such as transferrin, but notlimited to such; chemical elements and agents; devices, systems, and ormechanisms; liposomes, including liposomes conjugated with transferrin,but not limited to such; conformationally-constrained peptide drugstargeted at the blood-brain barrier; endogenous blood brain barrier andor blood tumor capillary transporters; inhibiting and or modulatingblood brain barrier active efflux transporters; air and or other gasbubbles; blood brain barrier breaking and or disrupting elements andagents; blood brain barrier tight junction separating and or endocytoseselements and agents; vector-mediated delivery of opioid peptides to thebrain; brain drug delivery of peptides and protein drugs viavector-mediated transport at the blood brain barrier, neurotrophic,neuroprotective, and various peptides and drugs, and the like.

In another illustrative embodiment, one or more elements cross variousin vivo biological barriers, such as the transmucosal passage, and mayalso cross the blood-brain barrier (BBB) and the blood-cerebrospinalfluid (CSF) barrier for targeted and or non-targeted in vivo delivery ofCNS agents and elements. In one embodiment, one or more BBB-passingelements comprise small and or large molecule drugs.

Natural Clathrin, and in particular its ability to ‘track’ vesicleproteins leaving a synapse into the extracellular space (Granseth, et al2007) indicates that the protein is not immediately scavenged by phagesand other “housecleaning” elements in the brain, and further, may movefreely about CNS spaces. In one embodiment, one or more elementsefficaciously move through the CNS spaces and comprise in situ elementsfor remediation, removal, and or sequestration of one or more types ofcontaminants, toxic elements, undesirable organic or inorganic elements,and the like.

In another embodiment, extensive modification and functionalization ofagents and elements may not be required for CNS entrance and or BBBpassage. Only minimal functionalization may be required, depending oncargo and element type.

In another embodiment, one or more CNS-entering and or BBB-passingelements of one or more types may behave as a drug by themselves—i.e.,they efficaciously operate alone without carrying additional elements,e.g., cargo elements. In another embodiment, one or more elements of oneor more types carry one or more additional elements of one more typespast the BBB.

In another illustrative embodiment, one or more elements enter the CNSand or cross the blood brain barrier for targeted delivery of agents andelements, including, but not limited to, small and or large molecules,non-lipid-soluble micromolecules, macromolecules, light sources,hydrophilic and or hydrophobic agents, such as therapeutic, diagnostic,and prosthetic agents, and other structured cargo to specific cells andareas within the brain, and such agents and or cargo may comprise one ormore sensor agents, assay agents, diagnostic agents, prosthetic agents,and also may comprise agents like central nervous system drugs,antibiotics, and antineoplastic agents of one or more types, but are notlimited to such.

In another embodiment, one or more elements are capable of circumventingthe fluid-brain barriers by intracellular routes related to threeseparate and distinct endocytic processes. The three endocytic processesfrom the least to the most specific are fluid- or bulk-phaseendocytosis, adsorptive endocytosis, and receptor-mediated endocytosis.

There are several transport mechanisms and techniques known in the artto be involved in the uptake of nanoparticles by the brain across theBBB (Lockman et al. 2002, Begley, 2004, de Boer et al. 2007), one ormore of which may be utilized in one or more invention embodiments.These mechanisms and techniques include: simple diffusion of lipophilicmolecules, the BBB-specific influx transporters, including organic anionand cation transporters and transcytosis or endocytosis. In oneembodiment, one or more elements are internalized at the BBB by one ortwo different endocytosis mechanisms: receptor-mediated endocytosis(RME) and adsorptive-mediated endocytosis (AME). AME is triggered by anelectrostatic interaction between the positively charged moiety of thepeptide and the negatively charged region of the plasma membrane. Incontrast, RME is specific to certain peptides such as insulin andtransferrin.

In one embodiment, delivery through the blood-brain barrier of one ormore types of small or large molecule cargo elements, and or moleculeswith polar functional groups is accomplished via chimeric peptides. Thelatter are formed when a transportable vector, such as cationizedalbumin, lectins, or a receptor-specific monoclonal antibody, isconjugated to a therapeutic compound that is normally not transportedthrough the BBB. In one embodiment, conjugation of drugs to transportvectors is facilitated by, but not limited to, the use of avidin-biotintechnology. In another embodiment, chimeric peptides are not required topass through the blood-brain barrier, depending on cargo and elementtypes.

In another illustrative embodiment, one or more elements may be coatedwith one or more surfactants and or cosurfactants, including, but notlimited to, polysorbate 20, 40, 60 and 80, and or with one or more othermaterials and substances to cross various biological barriers, such asthe transmucosal passage, and also to overcome the blood-brain barrier(BBB), the transmucosal passage, and the blood-cerebrospinal fluidbarrier (CSG) for targeted delivery of agents and elementsnanoparticles. In another embodiment, surfactants and or cosurfactantsare not required to achieve such BBB-passing functionality, depending oncargo and element type. E.g., in the prior art, it has been shown thatusing such surfactants and co-surfactants can cause an immunogenicresponse.

In another illustrative embodiment, one or more elements may becationized to facilitate blood brain barrier passage. In anotherembodiment, cationization is not required to achieve such functionality,depending on cargo and element type.

In another illustrative embodiment, one or more elements cross the bloodbrain barrier due to disruption of the barrier by acoustic techniques,such as by using ultrasound.

In another embodiment, zonula occludens toxin and its eukaryoticanalogue, zonulin, (zot) are protein ligands attached to one or moreinvention elements. Zonulin, the natural ligand of the Zot targetreceptor, interacts with these cargo attachment elements at the bloodbrain barrier, unlocking the tight junctions (TJ) in the brain thatregulate the blood-brain barrier at that receptor. TJ-unlocking allowspassage of one or more elements through the BBB, and thereby enablesdelivery of small and large molecules, non-lipid-soluble micromolecules,macromolecules, light sources, and other structured cargo elements tothe brain. In another embodiment, Zonulin is not required to passthrough the blood-brain barrier, depending on cargo and element types.

Extracellular pathways circumventing the fluid-brain barriers in humansare comparable in the CNS of rodents and a subhuman primate. The mosthighly documented extracellular route is through the circumventricularorgans (e.g., median eminence, organum vasculosum of the laminaterminalis, subfornical organ, and area postrema), all of which comprisefenestrated capillaries and, therefore, lie outside the BBB. In oneembodiment, blood-borne macromolecules; specifically fluid-phasemolecules released by the invention; escaping fenestrated vesselssupplying the circumventricular organs move extracellularly intoadjacent brain areas located behind the BBB.

The potential intracellular and extracellular pathways that blood-bornesubstances carried within one or more elements may follow in variousembodiments for circumventing the fluid-brain barriers and entry to theCNS are therefore numerous, and various invention embodiments are usedas appropriate. One invention embodiment, for example, uses the nasalcavity as a route for delivery of one or more types of drugs and otheragents, especially for systemically acting drugs that are difficult todeliver via routes other than injection. Embodiments for the use of thenasal cavity for drug delivery also extend to circumventing the bloodbrain barrier. Drugs have been shown to reach the CNS from the nasalcavity by a direct transport across the olfactory region situated at theloft of the nasal cavity. It is the only site in the human body wherethe nervous system is in direct contact with the surroundingenvironment. In one embodiment, the nasal route would be important forrapid uptake of one or more types of drugs used in crisis treatments andmanagement, such as for acute pain, epilepsy, psychic agitation, and forone or more other types of centrally acting drugs where the pathway fromnose to brain provides a faster and more specific therapeutic effect.Furthermore, in another embodiment, the trigeminal nerve and, inanimals, the vomeronasal organ also connects the nasal cavity with thebrain tissue. One or more methods of nasal delivery to the CNS, whichmay also be used by the instant invention, but not limited to, aredescribed in Dhuria, et al, 2008; Ma et al, 2007; and Thorne et al.1995.

The nasal cavity has a relatively large absorptive surface area and thehigh vascularity of the nasal mucosa ensures that absorbed compounds arerapidly removed (Mainardes, et al 2006). In one embodiment, two routes,singly or in combination, are used via which one or more types ofmolecules are transported from the olfactory epithelium into the CNSand/or CSF. The first is the epithelial pathway, where one or more typesof compounds pass paracellularly across the olfactory epithelium intothe perineural spaces, crossing the cribriform plate and entering thesubarachnoid space filled with CSF. From here the molecules can diffuseinto the brain tissue or will be cleared by the CSF flow into thelymphatic vessels and subsequently into the systemic circulation. Thesecond embodiment utilizes the olfactory nerve pathway, where compoundsmay be internalized into the olfactory neurones and pass inside theneuron through the cribriform plate into the olfactory bulb. In anotherembodiment, it is possible that further transport into the brain canoccur by bridging the synapses between the neurons. After reaching thebrain tissue, the drugs are cleared either via the CSF flow or viaefflux pumps such as p-glycoprotein at the BBB into the systemiccirculation. Despite the potential of the nasal route, there are somefactors that limit the intranasal absorption of drugs. These barriersinclude the physical removal from the site of deposition in the nasalcavity by the mucociliary clearance mechanisms, enzymatic degradation inthe mucus layer and nasal epithelium and the low permeability of thenasal epithelium removed (Mainardes, et al 2006). Colloidal carrierssystems, such as nanoparticles and liposomes have demonstrated greatefficacy in increasing drug bioavailability via the nasal route (Illum,2002) In one invention embodiment, one or more elements comprise acolloidal carrier for enhanced nasal delivery of one or more elements,of one or more types.

Further, in one embodiment, it is possible to greatly improve the nasalabsorption of one or more types of drugs and other elements byadministering them in combination with an absorption enhancer thatpromotes the transport of the drug across the nasal membrane. Anotherinvention embodiment comprises a nasal drug-delivery system thatcombines an absorption enhancing activity with a bioadhesive effect,which increases the residence time of the formulation in the nasalcavity. In one embodiment, this method can be even more effective forimproving the nasal absorption of polar drugs. In one or moreembodiments, a wide range of absorption enhancer systems can beutilized. In another embodiment, depending on cargo and element types,minimal functionalization may be required to take advantage of nasalabsorption for efficacious passage to brain cells.

In another illustrative embodiment, one or more elements and in one ormore configurations comprise in vivo and or in vitro sensor systems,assay systems, therapeutic drugs and other suitable methods to dogenetic-based (trait-based) and or phenotype (state-based) drug dosing.In one embodiment, drugs are delivered at optimally effective and safedoses per each individual.

The invention, in one embodiment, provides for individual patientfactors such as genotype, phenotype, age, gender, ethnicity etc., to betaken into account by one or more elements and factored into dosing andadministration consideration. It has been demonstrated thatinter-individual response variability can be 40-fold or more withpractically all classes of psychotropic drugs. This makes it difficultto formulate rational guidelines for dosing and interpretation ofbiological parameters (such as plasma or serum drug concentrations) thatmight be associated with a therapeutic response. Although much remainsunknown, a number of factors have been characterized as importantdeterminants of patient-to-patient variability. These encompassgenetics, disease state, nutritional status, concurrent use of drugs,and other pharmacoactive substances, including demographic factors suchas age, gender, and ethnicity. Therefore, there is a requirement for invivo systems that analyze many of these factors and dynamically adjustdosing accordingly.

In one embodiment, one or more elements comprise one or morepersonalized medicine elements, and which elements' efficacy may beincreased, because responses arising from one or more individualvariability factors; such as, but not limited to, genotype, phenotype,disease state, metabolic state, nutritional status, consistant use ofdrugs, and other pharmacoactive substances, and also demographic factorssuch as age, and ethnicity; are factored into the elements, pre-deliveryand or post delivery. Side effect profiles may also be reduced via suchpersonalized medicine embodiments.

In one embodiment, one or more elements comprise one or more patenteddrugs; drugs that are about to go off patent; have already gone offpatent (generics); and or their active metabolites, and which drugs'efficacy may be beneficially altered and or enhanced by use of theinvention. These beneficial changes in the status of an existing drugmay be achieved by the invention in one or more embodiments, forexample, but not limited to: the ability to target specific areas in thebody; to pass the blood brain barrier; to cross over into cells andtheir organelles; to fuse with cell membranes; to gain access to thecytosol; to offer the benefits of low antigenicity or minimalimmunogenic effects; to modify, regulate, and or control cellularprocesses; to more efficiently and efficaciously carry drugs; and or todynamically and or statically adjust the drug's responses and dosagesarising from inter-individual variability due to one or more factors,such as, but not limited to, genotype, phenotype, disease state,metabolic state, nutritional status, consistant use of drugs, and otherpharmacoactive substances, and also demographic factors such as age,gender, and ethnicity of the patient. New patent filings for about to gooff patent drugs and drugs already off patent may be enabled by one ormore invention embodiments, such as affording increased drug efficacy,and or by enabling a better safety profile for the drug in question.

In various embodiments, the instant invention can carry one or moretypes of biomedical or healthcare elements, for example and withoutlimitation: one or more therapeutic elements; pharmaceutical elements;diagnostic elements; assay elements; cosmetic elements; agents fortreating one or more types of autoimmune diseases; agents for treatingone or more types of infectious diseases; biological elements;radioactive agents or nuclear medicine agents; contrast agents;nano-scale biosensors; restorative agents; regenerative agents; cell,tissue, organ or circulatory repair elements; drug discovery agents;drug designer agents; drug research and development agents; drugfabrication agents; drug control and regulation agents; drug modifieragents; targeted drug delivery agents; clinical drug trial agents;antibiotics; antibacterials; vaccines; antiviral and anti-parasiticdrugs; cytostatics; vitamins; proteins and peptides, including enzymes;hormones or other biological elements; prosthetic elements; intelligentnano-prostheses that supplement or enhance cell, tissue, or organfunctioning; surgical elements; magnetic iron oxide nanoparticles;nano-scale biosensors; assays; diagnostic systems or nano-devices for invivo delivery of targeted therapy to combat diseases, such as cancer andHIV, and the like, including other types and forms of drug elements forthe diagnosis, cure, mitigation, treatment, prevention of disease. Someor all such elements may operate under the control and influence ofvarious other elements and or methods and comprise another type ofinvention platform.

In another illustrative embodiment, one or more elements in whole or inpart, cure, mitigate, or treat one or more types of bodily injuries andinsults, including traumatic injury, blood clots, and the like, but notlimited to.

In one embodiment, nano-engineered scaffolds comprised of a plurality ofelements are able to support and promote cellular differentiation andgrowth in injured or degenerated regions.

In one illustrative embodiment, one or more elements comprise one ormore types of small and or large molecules and may utilize one or moremethods to enter the CNS and or cross the blood brain barrier, in wholeor in part, for delivery of one or more assay, diagnostic, therapeuticagents, and drugs, of one or more types, to cells and or targeted areaswithin the brain, like, for example: contrast agents; central nervoussystem drugs; antibiotics; antineoplastic agents, which may be used fortreating malignant brain tumors (primary and or metastasized, of one ormore types) or benign neoplasms; Parkinson's agents; Multiple Sclerosisagents; epilepsy agents; meningitis agents; Alzheimer's disease agents;HIV infection agents; memory agents; stroke agents; coma agents; and thelike; or comprise one or more psychotropic agents or therapies of one ormore types to study, diagnose, cure, mitigate, or treat of one or moretypes of mental health and illness, including, but not limited to,stress; anxiety; depression; mania; bipolar disorder; attention deficit(hyperactivity) disorder; panic attacks; phobias; addictions; anger;rage; suicidal thoughts and tendencies; substance abuse disorder; posttraumatic stress disorder; psychoses; mental retardation; autism;delirium symptoms; schizophrenia; neuroses; and or enhancing memory;cognition; cognitive functioning; the effects of cognitive therapy, andthe like; including other types and forms of drug elements for thediagnosis, cure, mitigation, treatment, or prevention of one or moretypes of CNS diseases. In another illustrative embodiment, one or moreelements enter the CNS, including crossing the blood brain barrier, inwhole or in part, to diagnose, cure, mitigate, or treat one or moretypes of CNS injuries and insults, including traumatic brain injury,blood clots, and the like, but not limited to.

In one embodiment, one or more elements promote neuroprotection bylimiting the damaging effects of free radicals generated after headinjury, a major factor contributing to neuropsychiatric degenerativedisorders (e.g., Alzheimer's).

In one embodiment, nano-engineered scaffolds comprised of a plurality ofelements are able to support and promote neuronal differentiation andgrowth in injured or degenerated brain regions.

In another illustrative embodiment, one or more elements comprise alight source, for use, for example, but not limited to, in aphotodynamic therapy (PDT) system for age related macular degeneracy(AMD).

Compounds such as drugs, amino acids, carbohydrates, proteins,nucleotide bases, hormones, pesticides and co-enzymes have beensuccessfully used in the prior art for the preparation of selectiverecognition matrices. A wide variety of print molecules have been usedin various imprinting protocols known in the art. Of all the imprintingstrategies known in the art, it has become evident that the use ofnon-covalent interactions between the print molecule and the functionalmonomers is the more versatile. The apparent weakness of theseinteraction types, when considered individually, may be overcome byallowing a multitude of interaction points simultaneously. Together withthe fast association and dissociation kinetics of these bond types, sothat in a short time many possible combinations can be checked beforethe correct partners associate, this protocol has proven advantageous.Furthermore, the use of non-covalent interactions in the imprinting stepclosely resembles the recognition pattern observed in nature. Exampleinvention molecular imprint embodiments in the art include, but are notlimited to:

-   -   Fragmented polymer monoliths    -   Composite polymer beads    -   Polymer beads from suspension, emulsion or dispersion        polymerization    -   In-situ polymerization    -   Polymer particles bound in thin layers    -   Polymer membranes    -   Surface-imprinted polymer phases

In one illustrative embodiment, the invention uses molecular-imprinttechnology, wherein biodegradable films are used as a pliable templatefor elements, which elements are pressed into a film and then removed,leaving a physical mold of the element's shape. In one embodiment, thiscan facilitate catalysis of certain reactions and may also be used forshape selective separations. In other embodiments, imprinted polymersmay facilitate the fabrication of elements to achieve selectivediffusion; as chromatographic supports for the separation of enantiomersand oligonucleotides by invention elements; to provide the recognitionelement for an invention chemical sensor; and for the synthesis ofpolymeric materials that mimic biological cargo attachment elements andare targeted by invention elements, and or play a role in the design ofnew drugs. In one embodiment, this invention process provides forimprinted biodegradable capsule production with target or site-specificfeature sizes at the molecular level. Other invention embodiments mayutilize imprinted membranes and thin films that also function as anartificial cell wall for the selective transport of targeted drugs,peptides and biologically important molecules.

Surface imprinting involves the following steps: The print molecule,usually a large one, is first allowed to form adducts with functionalmonomers in solution and the formed elements are subsequently allowed tobind to an activated surface such as silica wafers or glass surfaces.Thus, with this technique, a designed imprinted (imaged) surface isobtained. This approach should potentially be valuable for creatingspecific cell binding surfaces. When preparing molecularly imprintedpolymer monoliths against large imprint species, there is a risk ofpermanent entrapment of the template in the polymer afterpolymerization. When using thin polymeric layers or imprinted surfacesthis drawback may be overcome.

In one embodiment, imprinted nanocapsules using techniques known in theart and as discussed above, one or more elements utilize and orconstitute a nanocapsule with manifold, multi-tiered capabilities for invivo administration and targeted delivery. The imprinted nanocapsule isdelivered in vivo to detect and target a particular in vitro imprintedbiological element, which may be, but is not limited to, a particulartype of receptor, protein, or cell, since its imprint shape on thenanocapsule will only bind in vivo to that particular biological elementtarget. The molecular-level imprint process thereby provides fortargeting one or more elements using biodegradable nanocapsules for invivo agent delivery. In addition, vectors and targeting moieties, andblood brain barrier, transmucosal, and CSF barrier breaching elements,and other elements and substances may also be attached to the surface ofthe molecular imprint nanocapsule or otherwise be conjugated to it.

In another illustrative embodiment, one or more elements may be used inconjunction with molecularly imprinted polymers known in the art asrecognition elements in biosensor-like devices. In one embodiment,imprinted polymer embodiments may be highly resistant sensing elementalternatives.

In another illustrative embodiment, one or more elements areencapsulated in whole or in part in one or more biodegradablecontrolled-release polymers, which polymers may also be conjugated withother elements and agents. The polymer capsule, and or one or moreelements may also be coated with one or more surfactants and orcosurfactants and or with other materials and substances. One or moretargeting and or masking moieties and or other targeting vectors mayalso be attached on the polymer surface, and or on one or more elements.

In one embodiment, one or more elements are put into one or morebiodegradable controlled-release polymeric capsules, and these elementstransform “dumb” polymeric delivery capsules into “smart” systems.

In the instance of polymeric nanocapsules, which may be molecularimprinted or not, illustrative controlled-release polymeric nanocapsuleembodiments of the invention may include one or more of the followingdelivery systems, but not limited to, and in one or more configurations:

1. Diffusion-controlled systems2. Water penetration-controlled delivery devices3. Chemically controlled systems4. Drugs covalently attached to polymer backbone systems, which deliverysystems can be further subdivided into soluble systems and insolublesystems. Insoluble systems are used as a subcutaneous or intramuscularimplant for the controlled release of the chemically tetheredtherapeutic agent. Soluble systems are used in targeting applications.5. Drug release determined predominantly by erosion systems, wherebycertain polymers can undergo a hydrolysis reaction at decreasing ratesfrom the surface of a device inward, and under special circumstances thereaction can be largely confined to the outer layers of a solid device.Two such polymers are poly (ortho esters) and polyanhydrides, becausethe rates of hydrolysis of these polymers can be varied within very widelimits, considerable control over the rate of drug release can beachieved.6. Poly (ortho esters) systems, which are highly hydrophobic polymersthat comprise acid-sensitive linkages in the polymer backbone.7. Polyanhydrides materials as bioerodible matrices for the controlledrelease of therapeutic agents. Aliphatic polyanhydrides hydrolyze veryrapidly while aromatic polyanhydrides hydrolyze very slowly, andexcellent control and regulate over the hydrolysis rate can be achievedby using copolymers of aliphatic and aromatic polyanhydrides. In thisway, erosion rates over many days have been demonstrated, and erosionsrates measured in years have been projected.

The form in which the foreign moiety, vector and or cargo are heldwithin one or more elements will depend on the release properties andmethods required. For release at the targeted site, it will be importantto ensure that the right conditions prevail, for example, to permit celllocalization and internalization via receptor mediated endocytosis.

In one illustrative embodiment, the invention enables one or more typesof delivery systems that engage in an iterative, interactive, anddynamic dialog with one or more targets; follow a sequence of actionsgoverned by biological control laws and methods; and or use behaviorsand methods as defined by graphs and or an algebra, for example, a Liealgebra. In one illustrative example, one or more elements follow analgorithm expressed by the invention, such as in this illustrativeembodiment:

-   -   1) One or more elements, that may be with or without cargo        elements, docks and or loiters on or near one or more cell        membranes,    -   2) One or more elements enter one or more target cells, while        one or more other elements continue to loiter nearby or stay        docked at the cell membrane.    -   3) The docked and or loitering element elements wait for a time        period,    -   4) The targeted cell produces one or more reactions, for        example, manufactures and or secretes an agent in response to        the element's docking and or delivering its cargo,    -   5) The docked element and or loitering elements analyze the new        cell behavior and or its secretions,    -   6) The docked element or loitering elements undergo a        conformational change in response to the cell's new behavior,    -   7) The docked element and or loitering elements self-adapt,        producing yet another conformational change in the cell, and or        releases another round of one or more agents that are taken up        by the targeted cell, and,    -   8) The foregoing process is repeated as required to achieve an        efficacious effect.

In another embodiment, one or more light sources comprised of one ormore elements operate in an intelligently staged sequence ororchestrated series of actions, which may be multiplexed or done inparallel by using one or more light and thermal energy emitting sourcesand methods. By using one or more light and or thermal energy emittingsources, optical and or thermal energies from one or more light sourcesoperate on one or more photosensitive and or thermal sensitive elementscomprising one or more elements that also comprise one or more entrappedagents. This method results in a staged series of overall actions thatfollow an intelligently ordered sequence of events. In an exampleembodiment, first a diagnostic agent from one or more elements isreleased by an optical and or thermal trigger, and the agent's positivefinding of a disease, like cancer or HIV then causes one or moretherapeutic agents to be released from the same and or other one or moreother elements by one or more optical and or thermal triggers. Agentdosages are released in calculated amounts, and the dosages may benon-targeted or targeted.

In another illustrative embodiment, cavity-forming cargo elements haveone or more compartments that in whole or in part are separated by oneor more barriers, for example, but not limited to, one or morephospholipid membrane barriers and or one or more barriers comprised ofmolecular-imprinted films. The barriers may exhibit structuraltransitions due to internal or external stimuli. In one embodiment,agents or cargo entrapped within one or more elements remain sequesteredwithin their respective compartments until a change in barrierpermeability state is triggered by contact, for example, by a ligand,with one or more specific targets or sites. The subsequent biochemicaland or biological reactions cause the barriers to alter states into anopened state and release entrapped cargo and agents from one or moreinvention elements. In one example embodiment, binary mixtures oftherapeutic and or diagnostic agents are mixed together as needed todynamically and more efficaciously deal with a disease or disorder.

The invention, in one or more embodiments, comprises in whole or in partone or more elements, components, devices, systems, and the like, of oneor more types, formed by using one or more engineering disciplines andrelated engineering technology disciplines of one or more types. Listedbelow are some such example invention embodiments, but are not limitedto.

In one embodiment, the invention remedies the deficiencies of prior artby providing one or more elements of one or more types, a plurality ofwhich may also comprise one or more nanoscale platforms of one or moretypes. A platform according to the invention may be used, for example,in biomedical, electronics, telecommunications, and informationprocessing applications.

FIG. 6 is an exemplary energy level diagram 600 illustrating the energylevels associated with a hyperfine interaction between electron andnuclear spin in the presence of magnetic fields of the type used to doESR spin label studies, which may be done in vivo and in vitro in oneinvention embodiment. The hyperfine interaction is a strictly quantummechanical phenomenon. In an atom, the electron possesses an intrinsicquantum mechanical quantity known as spin. The nucleus of an atom alsopossesses spin. Intrinsic spin tends to generate a spin magnetic momentthat is capable of interacting with other magnetic moments and fields.Generally, the spin magnetic moment of the nucleus does not interactwith the spin magnetic moment of the electron. However, in the presenceof a strong magnetic field, the spin magnetic moments of the electronand nucleus become coupled and interact.

In one illustrative embodiment, the electron is excited using pulses ofelectromagnetic radiation while maintaining its spin configuration. Thesource of the electromagnetic radiation may be, for example, an ordinarylamp, an LED, a time-varying magnetic field generator, a laser, or anelectromagnetic field generator. A hyperfine interaction gives rise toelectron nuclear double resonance (ENDOR) techniques. According to oneillustrative embodiment of the invention, room temperature EPR and ENDORtechniques known in the art are used for performing in vivo spin probestudies.

In another embodiment, one or more elements comprise one or morediagnostic agents, and during the same NMR/MRI, or EPR, or ESR, orESEEM, or ENDOR, or PET, or SPECT, or OCT operation, one or moreelements use quantum information processing techniques known in the artcan modify, process, manipulate, encode and decode, input, output,transmit, communicate, store and read information using one or moremodulated signals, methodologies, or carrier signals of one or moretypes.

In one embodiment, one or more invention elements in one or moreconfigurations, are bonded, tethered, or otherwise incorporated into oneor more invention and or non-invention elements, comprisingfunctionalized nanoscale elements, components, devices, systems, and orplatforms such as, but not limited to, nano-lasers, quantum dots;photonic dots; nanoscale DNA chips; protein assay chips; assay elements;environmental, protein, phenotype, DNA, and or metabolic assay andanalysis elements.

In another embodiment, one or more elements may comprise a bio-lasingstructure, in vivo or in vitro.

In one embodiment, one or more elements in one or more configurationscomprise nano-sensor elements; including, but not limited to,radioactivity sensors; chemical sensors; biological sensors;electromagnetic sensors; acoustic sensors; visible, infrared, and orultraviolet wavelength sensors; tactile sensors; pressure sensors;volumetric sensors; flow sensors; and temperature sensors; and one ormore of which sensors may constitute a bio-molecular device.

In one embodiment, one or more elements and or platforms utilize and oremploy one or more types of transmitter and or receiver elements assensors and or for transmission of information of one or more types invivo and in vitro.

In another embodiment, one or more elements and in one or moreconfigurations comprise one or more nanoscale elements, components,devices, systems, and or platforms that input, read out, process,analyze, output and report on information gathered by one or more typesof diagnostic, test, label, tag, reporter, sensor, and or assayelements.

In one embodiment, quantum dots and or photonic dots are released invivo or in vitro from one or more elements, and the quantum dots and orphotonic dots are coated in whole or in part in one or more surfactants,cosurfactants, and other materials or sequestering substances.

In one embodiment, quantum dots are tagged to one or more elements. Thespecific wavelength glow of the quantum dots enables the identificationof specific pathologies, disorders, metabolic states, proteins or DNAmaking it possible to diagnose various diseases.

In one embodiment, one or more nanoscale quantum dot assays using tinypermutations of color tag a million or more different proteins orgenetic sequences in a process called multiplexing. In one embodiment,one or more quantum dots of various sizes are excited at the samewavelength but have different emission wavelengths, and act as probes inexperiments where multiple fluorescent measurements need to be madesimultaneously, such as flow cytometry or confocal microscopy.

In another illustrative embodiment, one or more elements are sufficientto implement in vivo or in vitro genetic and protein nanoscale opticalbiological assay systems and methods. In one illustrative configuration,one or more elements comprise one or more nano-scale DNA chips known inthe art, and or one or more nano-scale DNA chips known in the art todetect DNA samples formed from bonding with the target DNA on a chip,and or reference DNA nano-chips.

In another illustrative configuration, one or more elements comprise oneor more protein array techniques known in the art. The array surfacesare designed to bind to one or more hydrophobic, hydrophilic (cation oranion) or specific ligands, and also include a protein array readerknown in the art.

In another illustrative embodiment, one or more elements are used in amultiplexed analysis system or method that provides a nanoscalereplacement for DNA-chip technology and can be used for the analysis ofgenetic variance, proteomics, and gene expression.

In another embodiment, one or more elements produce specific lightemissions and or thermal energies caused by their coming into contactwith a particular metabolic state, medical disorder, disease pathology,genotype, phenotype and or other specific stimuli. One or more entrappedagents carried by one or more elements are thereby selectively triggeredand released. In doing so, they form a targeted agent delivery systemwithout exposing the entire body—or an indiscriminate area—to a similardose of light, thermal energy, and or agents. The agents may bedelivered in vivo by means known in the art.

In one illustrative embodiment, photonic energies from one or moreelements thermally operate on one or more other elements that may haveone or more entrapped materials, such as, but not limited to,therapeutic, diagnostic, and or therapeutic agents within an aqueousinterior, and or that may have one or more entrapped nanoparticles suchas liposomes, micelles, proteins, other biological and or bioengineeredelements, including organic, inorganic, and synthetic materials, and orthat may have one or more hydrophobic materials bound to a lipid bilayermembrane. The well-known permeability increase at the phase transitiontemperature provides a means to trigger release of an entrapped agent,like, for example release of a therapeutic agent in locally heatedtissues. In one embodiment, efficient in vivo or in vitro release ofentrapped agents at non-targeted and or targeted sites are triggered bylight emitted by one or more light sources when the one or more elementscomprise a photoisomerisable species.

In another embodiment, the method of one or more LuxR proteins and luxbioluminescence genes and or other luminescent causing genes known inthe art are utilized and are bioengineered and incorporated into one ormore elements, ligands, targeting moieties, and or vectors, which mayalso be conjugated with one or more other elements, materials, andsubstances. In one embodiment, luminescent causing genes provide opticalpumping sufficient to excite one or more quantum dots and or photonicdots.

In an illustrative embodiment, in vivo release from one or more cargoelements comprised of one or more entrapped liposomal and ornon-liposomal-entrapped agents are optically triggered by photonsemitted by light sources of one or more types. In one illustrativeembodiment, one or more light sources produce specific light wavelengthemissions caused by their coming into contact with, for example, aspecific disease at in vivo target site and causes diagnostic,therapeutic, and or prosthetic agents comprised in a photosensitiveinvention delivery system to be triggered and released from one or moreinvention elements, thereby forming a highly targeted drug deliverysystem. For example, in one embodiment, one or more cargo elementscomprise an amphipathic lipid, such as a phospholipid, having two chainsderived from fatty acid that allow the lipid to pack into a bilayerstructure. One or more photosensitizers may be incorporated into theentrapped materials' cavity and or membranes.

In one illustrative embodiment, a phospholipid(1,2-(4′-n-butylphenyl)azo-4″(-phenylbutyroyl))-glycero-3-phosphocholine(‘Bis-Azo PC’), is substituted with azobenzene moieties in both acylchains that can be photoisomerised by a fast nanolaser pulse. One ormore other photoisomerisable species can be used in other embodiments.Agent release from one or more cargo elements occurs on the millisecondstimescale and photosensitised cargo elements thereby serve as lightsensitive elements to allow for the triggered release of agents from oneor more invention elements. In one embodiment, cholesterol additives maybe used. The addition of cholesterol may have a marked effect onkinetics of agent release from cargo elements, and in some circumstancescan result in substantial enhancement of light sensitivity in one ormore photosensitised elements comprising one or more invention elements,In another embodiment, thermal and photosensitive activation systemsacting together comprise one or more elements.

The invention, in one embodiment, comprises an in vitro and or in vivonanoscale, biomolecular electronics element and or nano-electronicselement, i.e., bio-molecular devices, which may be employed in ascalable, intelligent, biomolecular electronics device platform and or anano-electronics device platform. The platform may also be comprised ofone or more non-invention elements and devices, such as crystals,conductors, insulators, semiconductors, MEMS, and circuits, but notlimited to such. And further, the platform may also be coated in one ormore surfactants and or cosurfactants and or metals, elements, materialsand substances.

In one embodiment, one or more elements and or platforms are used forbiomolecular electronic and or nano-electronic devices. Biologicalmolecules, particularly proteins and lipids are used to perform thebasic properties necessary for the functioning of biomolecularelectronic devices. These biological materials conduct and transfermolecules from one location to another, are capable of major colorchanges on application of an electric field or light and can producecascades that can be used for amplification of an optical or anelectronic signal. All these properties can be applied to electronicswitches, gates, storage devices, biosensors, biological transistors, toname just a few. In general, the electrical properties of bilayer lipidmembranes are easily measurable for signal generation and transduction.In one embodiment, hybrid elements comprising cells with intact plasmamembranes can be considered to act as tiny capacitors under theinfluence of an electric field. Whereas sufficiently high field strengthmay increase the membrane potential past a critical point leading to thebreakdown of the membrane, experimental care must be taken. (Dielectricbreakdown of biological membrane occurs at about 1 volt across themembrane.) On the other hand, the use of electrostatic potentials aroundthe lipid molecules is very attractive, because they are controllable.

In one embodiment, one or more elements comprise nanoscale elements,components, devices, systems and or platforms, in one or moreconfigurations, which form connectors for carrying information from astorage, processing or communications element or device to another, ofone or more types.

In one embodiment, one or more elements comprise one or more informationprocessing elements, components, devices, systems and or platforms suchas, for example, but not limited to, encoders and decoders, memory,logic gates, registers, circuits, wiring and connectors, input andoutput elements, analog to digital and digital to analog converters andsystem architectures known in the art.

In one embodiment, one or more invention elements comprise nanoscaleelements, components, devices, systems and or platforms that modify,process, manipulate, encode and decode, input, output, transmit,communicate, store and read various forms and types of information usinga variety of suitable techniques known in the art, in vivo and in vitro.

A scalable information-processing invention platform may also include anencoder, e.g., a predetermined or specific DNA sequence thatdeliberately encodes at least a subset of the elements to take the formof specified sequence, as well as a decoder for reading information fromat least a subset of the protein-based information processing elements.Examples of such a bio-system decoder are, but not limited to, adye-based protein assay, a quantum dot-based assay, or other proteinassay methods known in the art. Another example of encoders/decoders isthe use of NMR and ESR and other methods known in the art that caneffect and discern protein behaviors and their physical characteristics.Another example of encoders/decoders is the use of photons of differentwavelengths and photo detectors.

In one embodiment, one or more elements comprise in vitro and or in vivonanoscale information processing elements, components, devices, systemsand or platform, which may follow and execute algorithms of one or moretypes expressed by or use biological control and regulate laws,processes, and or methods, and or geometrically derived algorithms suchas graphs and Lie algebras, including Clifford algebras, but not limitedto.

In another embodiment, one or more elements comprise a cognitiveinformation processing element, device, and or platform of one or moretypes that follow and execute algorithms expressed by or use biologicalcontrol and regulate laws and or processes, and or geometrically derivedalgorithms such as graphs and Lie algebras, including Clifford algebras,but not limited to.

In another embodiment, one or more elements comprise a hybrid digitaland analog information processing element, device, and or platform ofone or more types, wherein enlisting the rich repertoire of biochemicalreactions and adopting a nested hierarchical organization makesintermixing of digital an analog processing possible in bio-computingapplications.

In one embodiment, one or more elements comprise one or more nanoscaleinformation processing elements, components, devices, systems and orplatform that utilize photons emitted by invention light sources of oneor more types as the basis of computation and or transmission andcommunication.

According to one illustrative embodiment, one or more elements compriseone or more nano-computer elements, components, devices, systems and orplatforms of one or more types that are programmable, and or autonomousacting, and or do cognitive processing, which bio-nano-computers mayalso utilize self-replicating, self-adapting, self-repairing,self-regulating, and or self-regenerating methods, and which are usedfor applications at the cellular, molecular, and nanoscale level thatmay include, but are not limited to, biomedical imaging, sensors,diagnostic systems, assay systems, therapeutic systems, drug deliverysystems, prosthetic systems, cybernetic systems, cellular-levelnano-fabrication systems, and inter- and intra-cellular imaging, repair,and engineering systems, the monitoring, sensing, imaging, diagnosing,repairing, constructing, fabricating, and or control and regulating oforganic and or inorganic elements, and which bio-nano-computer elementsand or platforms also may utilize and leverage biological control andregulate laws and or methods, and or geometrically derived algorithmssuch as graphs and Lie algebras, including Clifford algebras, but notlimited to, in the performance of their tasks.

In one illustrative embodiment, one or more element chains are createdvia a molecular bridge group. To align the elements with respect to oneanother and also with respect to an external magnetic or electricalfield. In one embodiment, one or more elements and or platforms and inone or more configurations are embedded in another material, like liquidcrystal.

In one embodiment, one or more elements and or platforms and in one ormore configurations are coated completely and or partially in a metal.

In another embodiment, one or more elements and or platforms and in oneor more configurations are coated completely and or partially inreflective and or non-reflective coatings.

In one embodiment, one or more elements and or platforms and in one ormore configurations are used to coat completely and or partially metals,crystals, insulators, conductors, semiconductor components, wires, anddevices.

In another illustrative embodiment, one or more elements and orplatforms and in one or more configurations facilitate the externallyand or mechanistically directed alignment of, for example, but notlimited to, biological elements, various other non-inventionnanoparticles, carbon nanotubes, crystals, conductors, semiconductors,insulators, and or other devices, materials and substances, whichaligned assemblies may further be coated in one or more surfactants andor metals, elements, materials and substances.

In one embodiment, one or more elements in one or more configurationsinclude other types of nanoparticle elements such as, but not limitedto, polymer-based, polybutylcyanoacrylate-based, and cetyl alcohol-basednanoparticles, empty cage Fullerenes, endohedral Fullerenes, carbonnanotubes, cells, liposomes, capsids, dendrimers, micelles, and thelike.

In another illustrative embodiment, one or more elements and orplatforms of one or more types in whole or in part enable a shapeprogrammable and or scaffolding system to which one or elements of oneor more types, including natural and or non-invention elements areaffixed and or further form more one or more structures of one moretypes

In one embodiment, one or more elements and or platforms in one or moreconfigurations form and or include optical elements such as, but notlimited to, optics; optoelectronic elements; photoelectric elements;photodetectors; and photosensitive elements, which optical elements mayalso be coated or treated in whole or in part with materials that affecttheir optical properties.

In one embodiment, one or more elements and or platforms and in one ormore configurations form and or include imaging elements and sensors,such as, but not limited to, CCDs and CMOS optical elements.

In one embodiment, one or more elements and or platforms, in one or moreconfigurations include and or comprise photonic to electrical energyconversion elements.

In one embodiment, one or more elements and or platforms form one ormore electronic circuits, which circuit may also be comprised of one ormore other elements such as empty Fullerenes, endohedral Fullerenes,nanotubes, crystals, insulators, conductors, semiconductors, and orother materials, substances and devices, which circuits also may becoated in one or more surfactants and or cosurfactants and or othermaterials and substances.

In one embodiment, one or more elements and or platforms are switched onor off and or change states by applying an electric field, and may alsocomprise one or more transistors or devices in another embodiment.

In another embodiment, one or more elements and or platforms and in oneor more configurations; self-assemble, and or are shape-programmed, andor use biological control and regulate laws, processes and methods, andor use geometrically derived algorithms such as graphs and Lie algebras,including Clifford algebras, but not limited to, and or are mechanicallyassembled via lithography, and or utilize other externally directedtechniques and methods known the art, and or some combination thereof;form natural positions that are associated with electronic circuits andor information processing devices, such as atomic and molecular scaledevice design, their interconnection, nanofabrication and circuitarchitectures.

According to one illustrative embodiment, one or more elements and orplatforms comprise one or more crystal structures and elements, of oneor more types.

According to one illustrative embodiment, one or more elements and orplatforms comprise one or more desiccated elements, of one or moretypes.

According to one illustrative embodiment, one or more invention compriseone or more hydrated and or rehydrated elements and or platforms, of oneor more types.

According to one illustrative embodiment, one or more elements and orplatforms comprise one or more rehydration elements and or platforms, ofone or more types.

According to one illustrative embodiment, one or more elements and orplatforms are embedded and or incorporated into one or more materials,substances, devices, agents, devices, systems, organisms, and ormechanisms of one or more types.

In another illustrative embodiment, one or more elements and orplatforms comprise one or more magnetic nanoparticles of one or moretypes.

In one embodiment, one or more elements and or platforms are nanoscalerecording memory media or components, which may incorporate metals,ferromagnetic materials, and or ferroelectric materials and elements,and or may form into magnetic rings, and or may form verticallypolarized magnetic domains and or form magnetic domains on isolatedislands of one or more types.

In one embodiment, one or more elements and or platforms are nanoscalephotovoltaic cells or components of one or more types.

In one embodiment, one or more elements are nanoscale batteries orcomponents of one or more type for storing electronic charge.

In one embodiment, one or more elements and or platforms comprise ananoscale environmental hazard-screening device, and or comprise an insitu remediation, removal and or sequestration component or system ofone or more types.

In one embodiment, one or more elements and or platforms comprise anopto-electronic device, system or component of one or more types.

In one illustrative embodiment, embodiment, one or more elementscomprise one or more nanoscale passive and or active linear or nonlinearoptic components, and or particle detectors, and or other elementssufficient to implement in vivo or in vitro optical system arrays andmethods.

In another embodiment, one or more elements comprise in vivo or in vitrodetection, diagnostic and tracking agents for chemical, biological, andor nuclear elements and activities, but not limited to such.

In one embodiment, one or more elements and or platforms comprise aspin-based electronics element or system of one or more types.

In one embodiment, one or more elements and or platforms exploit theCoulomb blockade-like properties of self-assembled proteins, wherein asingle particle at a time may move through a transmembrane protein-basedchannel.

In one embodiment, one or more elements and or platforms utilize and orexploit the Casimir effect, which is a small attractive force that actsbetween two closely parallel, uncharged conducting elements. It is dueto quantum vacuum fluctuations of the electromagnetic field.

In some illustrative embodiments, one or more elements and or platformsand in one or more configurations are physically linked via molecularaddends of one or more types, but are not limited to such addend types.

In other illustrative configurations, one or more elements and orplatforms are functionally linked via photonic, chemical,electromagnetic, electrical and/or quantum (non-classical) interactionsof one or more types, including the Internet, to work and cooperatelocally and/or remotely.

One or more elements and or platforms of one or more types may beencapsulated, packaged, stored, incorporated, and or utilize one or moremethods known in the art, including for example, but not limited to:catheters; injections, including intramuscular injections; syringes;droppers and bulbs; pills; intravenous means; oral means; anal means;capsules; nanocapsules; nanoparticles; nano-devices; prescriptions;hospital and medical supplies; dental supplies; non-prescriptions;medications; over the counter products and remedies; alternativemedicine supplies, systems, products and devices; hair care products;splints, casts, walkers, crutches, canes, wheelchairs, and otherambulatory aids; natural foods; vitamin and mineral supplements; firstaid products; emergency health care procedures, systems, devices, andproducts, including combat medicine; health care products; grafts; skinpatches; bandages; adhesives; wraps; masks; markers; powders; granules;geriatric care products; pediatric care products; diagnostic devices,systems, and products; medical imaging devices, systems, and products;telemedicine devices, systems, and products; in vivo monitoring systems,products, systems, and devices; in vitro monitoring systems, products,systems, and devices; laundry products; chemical, nuclear and biologicalsensors; sensors; bio-sensors; environmental sensors; combat systems,clothing, uniforms, and protective gear; food preparation products; foodtesting and safety devices, systems, and products; food storage wraps,systems, devices, and products; water treatment devices, systems andproducts; waste storage, management, and treatment systems and products;sewerage systems and products; plumbing systems and products; bed andbath products; animal care and veterinary products; animal feed; animalslaughter systems and products; cooking products; cookware; forensicdevices, systems and products; home and office cleaning products; homeproducts; office products; personal products; industrial products; homeand office care products; paper products; personal hygiene products;sexual hygiene and safety products; sexual reproduction devices,systems, and products; sexual arousal products and devices; dental anddental care products; oral hygiene products, devices, and systems;robotic products, systems and devices; cybernetic devices; jewelry;novelties; solvents; agro-pro ducts; plants; animals; vehicles;biologicals; chemicals; cells; tissue; organs; proteins; liposomes;phages; micelles; peptides; antibodies; monoclonal antibodies; DNA; RNA;IRNA; siRNA; RISC; cloning; human contact; micro-electromechanicalsystems (MEMS) and other types of nano-systems; food utensils; tools;appliances; consumer electronics; paints and finishes; heating,ventilation and air conditioning systems; construction, building, homeand office materials; water; milk; food and other edible or chewablesubstances and items; prostheses; food and drink additives andsupplements; drinks; beverages; soaps; creams; ointments; salves;topical agents; cosmetics; beautifying agents; liquids; fluids; oils;gels; adhesives; aerosols; vapors; airborne methods; pumps; fragrancesand perfumes; textiles; sporting and athletic goods and devices;physical work out and training systems, devices, and products; sportsmedicine systems, devices, and products; recreational products and gear;shoes, clothing, and apparel; eyewear; sprays; dyes; biologicalelements; organ transplants; implants; stents; prosthetic devices;artificial skin, blood, limbs, joints, bones, cells, eyes, organs, andother artificial body parts and biological elements; subcutaneous means;incisions; surgical means; and in-patient and out-patient medicalprocedures.

The above-described embodiments have been set forth to describe morecompletely and concretely the present invention, and are not to beconstrued as limiting the invention. It is further intended that allmatter and the description and drawings be interpreted as illustrativeand not in a limiting sense. That is, while various embodiments of theinvention have been described in detail, other alterations, which willbe apparent to those skilled in the prior art, are intended to beembraced within the spirit and scope of the invention.

In view of the foregoing, what is claimed is:

TABLE 1 Human protein: Clathrin heavy chain 1Source: http://www.expasy.ch/uniprot/Q00610#seqLength; 1675AA MW; 191615 Da [This is the MW of the unprocessedprecursor] Protein name; Clathrin heavy chain 1 Synonym; CHC-17Gene name: Name: CLTC Synonyms: CLH17, CLTCL2, KIAA0034Isoform 1 of Clathrin heavy chain 1 - Length: 1675 AS        10         20         30         40         50         60MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN        70         80         90        100        110        120PIRRPISADS AIMNPASKVI ALKAGKTLQI FNIEMKSKMK AHTMTDDVTF WKWISLNTVA       130        140        150        160        170        180LVTDNAVYHW SMEGESQPVK MFDRHSSLAG CQIINYRTDA KQKWLLLTGI SAQQNRVVGA       190        200        210        220        230        240MQLYSVDRKV SQPIEGHAAS FAQFKMEGNA EESTLFCFAV RGQAGGKLHI IEVGTPPTGN       250        260        270        280        290        300QPFPKKAVDV FFPPEAQNDF PVAMQISEKH DVVFLITKYG YIHLYDLETG TCIYMNRISG       310        320        330        340        350        360ETIFVTAPHE ATAGIIGVNR KGQVLSVCVE EENIIPYITN VLQNPDLALR MAVRNNLAGA       370        380        390        400        410        420EELFARKFNA LFAQGNYSEA AKVAANAPKG ILRTPDTIRR FQSVPAQPGQ TSPLLQYFGI       430        440        450        460        470        480LLDQGQLNKY ESLELCRPVL QQGRKQLLEK WLKEDKLECS EELGDLVKSV DPTLALSVYL       490        500        510        520        530        540RANVPNKVIQ CFAETGQVQK IVLYAKKVGY TPDWIFLLRN VMRISPDQGQ QFAQMLVQDE       550        560        570        580        590        600EPLADITQIV DVFMEYNLIQ QCTAFLLDAL KNNRPSEGPL QTRLLEMNLM HAPQVADAIL       610        620        630        640        650        660GNQMFTHYDR AHIAQLCEKA GLLQRALEHF TDLYDIKRAV VHTHLLNPEW LVNYFGSLSV       670        680        690        700        710        720EDSLECLRAM LSANIRQNLQ ICVQVASKYH EQLSTQSLIE LFESFKSFEG LFYFLGSIVN       730        740        750        760        770        780FSQDPDVHFK YIQAACKTGQ IKEVERICRE SNCYDPERVK NFLKEAKLTD QLPLIIVCDR       790        800        810        820        830        840FDFVHDLVLY LYRNNLQKYI EIYVQKVNPS RLPVVIGGLL DVDCSEDVIK NLILVVRGQF       850        860        870        880        890        900STDELVAEVE KRNRLKLLLP WLEARIHEGC EEPATHNALA KIYIDSNNNP ERFLRENPYY       910        920        930        940        950        960DSRVVGKYCE KRDPHLACVA YERGQCDLEL INVCNENSLF KSLSRYLVRR KDPELWGSVL       970        980        990       1000       1010       1020LESNPYRRPL IDQVVQTALS ETQDPEEVSV TVKAFMTADL PNELIELLEK IVLDNSVFSE      1030       1040       1050       1060       1070       1080HRNLQNLLIL TAIKADRTRV MEYINRLDNY DAPDIANIAI SNELFEEAFA IFRKFDVNTS      1090       1100       1110       1120       1130       1140AVQVLIEHIG NLDRAYEFAE RCNEPAVWSQ LAKAQLQKGM VKEAIDSYIK ADDPSSYMEV      1150       1160       1170       1180       1190       1200VQAANTSGNW EELVKYLQMA RKKARESYVE TELIFALAKT NRLAELEEFI NGPNNAHIQQ      1210       1220       1230       1240       1250       1260VGDRCYDEKM YDAAKLLYNN VSNFGRLAST LVHLGEYQAA VDGARKANST RTWKEVCFAC      1270       1280       1290       1300       1310       1320VDGKEFRLAQ MCGLHIVVHA DELEELINYY QDRGYFEELI TMLEAALGLE RAHMGMFTEL      1330       1340       1350       1360       1370       1380AILYSKFKPQ KMREHLELFW SRVNIPKVLR AAEQAHLWAE LVFLYDKYEE YDNAIITMMN      1390       1400       1410       1420       1430       1440HPTDAWKEGQ FKDIITKVAN VELYYRAIQF YLEFKPLLLN DLLMVLSPRL DHTRAVNYFS      1450       1460       1470       1480       1490       1500KVKQLPLVKP YLRSVQNHNN KSVNESLNNL FITEEDYQAL RTSIDAYDNF DNISLAQRLE      1510       1520       1530       1540       1550       1560KHELIEFRRI AAYLFKGNNR WKQSVELCKK DSLYKDAMQY ASESKDTELA EELLQWFLQE      1570       1580       1590       1600       1610       1620EKRECFGACL FTCYDLLRPD VVLETAWRHN IMDFAMPYFI QVMKEYLTKV DKLDASESLR      1630       1640       1650       1660       1670KEEEQATETQ PIVYGQPQLM LTAGPSVAVP PQAPFGYGYT APPYGQPQPG FGYSM

TABLE 2 Human protein: P53675 - Clathrin heavy chain 2 (CHC-22)Length: 1640 aa, molecular weight: 187030 Dahttp://harvester.embl.de/harvester/P536/P53675.htmMAQILPVRFQ EHFQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV TIIDMSDPMA   60PIRRPISAES AIMNPASKVI ALKAGKTLQI FNIEMKSKMK AHTMAEEVIF WKWVSVNTVA  120LVTETAVYHW SMEGDSQPMK MFDRHTSLVG CQVIHYRTDE YQKWLLLVGI SAQQNRVVGA  180MQLYSVDRKV SQPIEGHAAA FAEFKMEGNA KPATLFCFAV RNPTGGKLHI IEVGQPAAGN  240QPFVKKAVDV FFPPEAQNDF PVAMQIGAKH GVIYLITKYG YLHLYDLESG VCICMNRISA  300DTIFVTAPHK PTSGIIGVNK KGQVLSVCVE EDNIVNYATN VLQNPDLGLR LAVRSNLAGA  360EKLFVRKFNT LFAQGSYAEA AKVAASAPKG ILRTRETVQK FQSIPAQSGQ ASPLLQYFGI  420LLDQGQLNKL ESLELCHLVL QQGRKQLLEK WLKEDKLECS EELGDLVKTT DPMLALSVYL  480RANVPSKVIQ CFAETGQFQK IVLYAKKVGY TPDWIFLLRG VMKISPEQGL QFSRMLVQDE  540EPLANISQIV DIFMENSLIQ QCTSFLLDAL KNNRPAEGLL QTWLLEMNLV HAPQVADAIL  600GNKMFTHYDR AHIAQLCEKA GLLQQALEHY TDLYDIKRAV VHTHLLNPEW LVNFFGSLSV  660EDSVECLHAM LSANIRQNLQ LCVQVASKYH EQLGTQALVE LFESFKSYKG LFYFLGSIVN  720FSQDPDVHLK YIQAACKTGQ IKEVERICRE SSCYNPERVK NFLKEAKLTD QLPLIIVCDR  780FGFVHDLVLY LYRNNLQRYI EIYVQKVNPS RTPAVIGGLL DVDCSEEVIK HLIMAVRGQF  840STDELVAEVE KRNRLKLLLP WLESQIQEGC EEPATHNALA KIYIDSNNSP ECFLRENAYY  900DSSVVGRYCE KRDPHLACVA YERGQCDLEL IKVCNENSLF KSEARYLVCR KDPELWAHVL  960EETNPSRRQL IDQVVQTALS ETRDPEEISV TVKAFMTADL PNELIELLEK IVLDNSVFSE 1020HRNLQNLLIL TAIKADRTRV MEYISRLDNY DALDIASIAV SSALYEEAFT VFHKFDMNAS 1080AIQVLIEHIG NLDRAYEFAE RCNEPAVWSQ LAQAQLQKDL VKEAINSYIR GDDPSSYLEV 1140VQSASRSNNW EDLVKFLQMA RKKGRESYIE TELIFALAKT SRVSELEDFI NGPNNAHIQQ 1200VGDRCYEEGM YEAAKLLYSN VSNFARLAST LVHLGEYQAA VDNSRKASST RTWKEVCFAC 1260MDGQEFRFAQ LCGLHIVIHA DELEELMCYY QDRGYFEELI LLLEAALGLE RAHMGMFTEL 1320AILYSKFKPQ KMLEHLELFW SRVNIPKVLR AAEQAHLWAE LVFLYDKYEE YDNAVLTMMS 1380HPTEAWKEGQ FKDIITKVAN VELCYRALQF YLDYKPLLIN DLLLVLSPRL DHTWTVSFFS 1440KAGQLPLVKP YLRSVQSHNN KSVNEALNHL LTEEEDYQGL RASIDAYDNF DNISLAQQLE 1500KHQLMEFRCI AAYLYKGNNW WAQSVELCKK DHLYKDAMQH AAESRDAELA QKLLQWFLEE 1560GKRECFAACL FTCYDLLRPD MVLELAWRHN LVDLAMPYFI QVMREYLSKV DKLDALESLR 1620KQEEHVTEPA PLVFDFDGHE 1640

TABLE 3 Human protein: P09496 - Clathrin light chain A (Lca).Length: 248 aa, molecular weight: 27077 DaSource: http://harvester.embl.de/harvester/P094/P09496.htmMAELDPFGAP AGAPGGPALG NGVAGAGEED PAAAFLAQQE SEIAGIENDE AFAILDGGAP  60GPQPHGEPPG GPDAVDGVMN GEYYQESNGP TDSYAAISQV DRLQSEPESI RKWREEQMER 120LEALDANSRK QEAEWKEKAI KELEEWYARQ DEQLQKTKAN NRVADEAFYK QPFADVIGYV 180TNINHPCYSL EQAAEEAFVN DIDESSPGTE WERVARLCDF NPKSSKQAKD VSRMRSVLIS 240LKQAPLVH 248

TABLE 4 Human protein: P09497 - Clathrin light chain B (Lcb)Length: 229 aa, molecular weight: 25190 Da,Source: http://harvester.embl.de/harvester/P094/P09497.htmMADDFGFFSS SESGAPEAAE EDPAAAFLAQ QESEIAGIEN DEGFGAPAGS HAAPAQPGPT  60SGAGSEDMGT TVNGDVFQEA NGPADGYAAI AQADRLTQEP ESIRKWREEQ RKRLQELDAA 120SKVTEQEWRE KAKKDLEEWN QRQSEQVEKN KINNRIADKA FYQQPDADII GYVASEEAFV 180KESKEETPGT EWEKVAQLCD FNPKSSKQCK DVSRLRSVLM SLKQTPLSR 229

TABLE 5Human protein: P53621 - Coatomer subunit alpha (Alpha-coat protein)Length: 1224 aa, molecular weight: 138332 Da, CRC64Source: http://harvester.embl.de/harvester/P536/P53621.htmMLTKFETKSA RVKGLSFHPK RPWILTSLHN GVIQLWDYRM CTLIDKFDEH DGPVRGIDFH   60KQQPLFVSGG DDYKIKVWNY KLRRCLFTLL GHLDYIRTTF FHHEYPWILS ASDDQTIRVW  120NWQSRTCVCV LTGHNHYVMC AQFHPTEDLV VSASLDQTVR VWDISGLRKK NLSPGAVESD  180VRGITGVDLF GTTDAVVKHV LEGHDRGVNW AAFHPTMPLI VSGADDRQVK IWRMNESKAW  240EVDTCRGHYN NVSCAVFHPR QELILSNSED KSIRVWDMSK RTGVQTFRRD HDRFWVLAAH  300PNLNLFAAGH DGGMIVFKLE RERPAYAVHG NMLHYVKDRF LRQLDFNSSK DVAVMQLRSG  360SKFPVFNMSY NPAENAVLLC TRASNLENST YDLYTIPKDA DSQNPDAPEG KRSSGLTAVW  420VARNRFAVLD RMHSLLIKNL KNEITKKVQV PNCDEIFYAG TGNLLLRDAD SITLFDVQQK  480RTLASVKISK VKYVIWSADM SHVALLAKHA IVICNRKLDA LCNIHENIRV KSGAWDESGV  540FIYTTSNHIK YAVTTGDHGI IRTLDLPIYV TRVKGNNVYC LDRECRPRVL TIDPTEFKFK  600LALINRKYDE VLHMVRNAKL VGQSIIAYLQ KKGYPEVALH FVKDEKTRFS LALECGNIEI  660ALEAAKALDD KNCWEKLGEV ALLQGNHQIV EMCYQRTKNF DKVSFLYLIT GNLEKLRKMM  720KIAEIRKDMS GHYQNALYLG DVSERVRILK NCGQKSLAYL TAATHGLDEE AESLKETFDP  780EKETIPDIDP NAKLLQPPAP IMPLDTNWPL LTVSKGFFEG TIASKGKGGA LAADIDIDTV  840GTEGWGEDAE LQLDEDGFVE ATEGLGDDAL GKGQEEGGGW DVEEDLELPP ELDISPGAAG  900GAEDGFFVPP TKGTSPTQIW CNNSQLPVDH ILAGSFETAM RLLHDQVGVI QFGPYKQLFL  960QTYARGRTTY QALPCLPSMY GYPNRNWKDA GLKNGVPAVG LKLNDLIQRL QLCYQLTTVG 1020KFEEAVEKFR SILLSVPLLV VDNKQEIAEA QQLITICREY IVGLSVETER KKLPKETLEQ 1080QKRICEMAAY FTHSNLQPVH MILVLRTALN LFFKLKNFKT AATFARRLLE LGPKPEVAQQ 1140TRKILSACEK NPTDAYQLNY DMHNPFDICA ASYRPIYRGK PVEKCPLSGA CYSPEFKGQI 1200CRVTTVTEIG KDVIGLRISP LQFR 1224

TABLE 6Human protein: P53618 - Coatomer subunit beta (Beta-coat protein)Length: 953 aa, molecular weight: 107139 Da, CRC64Source: http://harvester.embl.de/harvester/P536/P53618.htmMTAAENVCYT LINVPMDSEP PSEISLKNDL EKGDVKSKTE ALKKVIIMIL NGEKLPGLLM  60TIIRFVLPLQ DHTIKKLLLV FWEIVPKTTP DGRLLHEMIL VCDAYRKDLQ HPNEFIRGST 120LRFLCKLKEA ELLEPLMPAI RACLEHRHSY VRRNAVLAIY TIYRNFEHLI PDAPELIHDF 180LVNEKDASCK RNAFMMLIHA DQDRALDYLS TCIDQVQTFG DILQLVIVEL IYKVCHANPS 240ERARFIRCIY NLLQSSSPAV KYEAAGTLVT LSSAPTAIKA AAQCYIDLII KESDNNVKLI 300VLDRLIELKE HPAHERVLQD LVMDILRVLS TPDLEVRKKT LQLALDLVSS RNVEELVIVL 360KKEVIKTNNV SEHEDTDKYR QLLVRTLHSC SVRFPDMAAN VIPVLMEFLS DNNEAAAADV 420LEFVREAIQR FDNLRMLIVE KMLEVFHAIK SVKIYRGALW ILGEYCSTKE DIQSVMTEIR 480RSLGEIPIVE SEIKKEAGEL KPEEEITVGP VQKLVTEMGT YATQSALSSS RPTKKEEDRP 540PLRGFLLDGD FFVAASLATT LTKIALRYVA LVQEKKKQNS FVAEAMLLMA TILHLGKSSL 600PKKPITDDDV DRISLCLKVL SECSPLMNDI FNKECRQSLS HMLSAKLEEE KLSQKKESEK 660RNVTVQPDDP ISFMQLTAKN EMNCKEDQFQ LSLLAAMGNT QRKEAADPLA SKLNKVTQLT 720GFSDPVYAEA YVHVNQYDIV LDVLVVNQTS DTLQNCTLEL ATLGDLKLVE KPSPLTLAPH 780DFANIKANVK VASTENGIIF GNIVYDVSGA ASDRNCVVLS DIHIEIMDYI QPATCTDAEF 840RQMWAELEWE NKVTVNTNMV DLNDYLQHIL KSTNMKCLTP EKALSGYCGF MAANLYARSI 900FGEDALANVS SEKPLHQGPD AAVTVHIRIR AKSQGMALSL GDKINLSQKE TSI 953

TABLE 7Human protein: P35606 - Coatomer subunit beta′ (Beta′-coat protein)Length: 905 aa, molecular weight: 102356 Da, CRC64Source: http://harvester.embl.de/harvester/P356/P35606.htmPLRLDIKRKL TARSDRVKSV DLHPTEPWML ASLYNGSVCV WNHETQTLVK TFEVCDLPVR  60AAKFVARKNW VVTGADDMQI RVFNYNTLER VHMFEAHSDY IRCIAVHPTQ PFILTSSDDM 120LIKLWDWDKK WSCSQVFEGH THYVMQIVIN PKDNNQFASA SLDRTIKVWQ LGSSSPNFTL 180EGHEKGVNCI DYYSGGDKPY LISGADDRLV KIWDYQNKTC VQTLEGHAQN VSCASFHPEL 240PIIITGSEDG TVRIWHSSTY RLESTLNYGM ERVWCVASLR GSNNVALGYD EGSIIVKLGR 300EEPAMSMDAN GKIIWAKHSE VQQANLKAMG DAEIKDGERL PLAVKDMGSC EIYPQTIQHN 360PNGRFVVVCG DGEYIIYTAM ALRNKSFGSA QEFAWAHDSS EYAIRESNSI VKIFKNFKEK 420KSFKPDFGAE SIYGGFLLGV RSVNGLAFYD WDNTELIRRI EIQPKHIFWS DSGELVCIAT 480EESFFILKYL SEKVLAAQET HEGVTEDGIE DAFEVLGEIQ EIVKTGLWVG DCFIYTSSVN 540RLNYYVGGEI VTIAHLDRTM YLLGYIPKDN RLYLGDKELN IISYSLLVSV LEYQTAVMRR 600DFSMADKVLP TIPKEQRTRV AHFLEKQGFK QQALTVSTDP EHRFELALQL GELKIAYQLA 660VEAESEQKWK QLAELAISKC QFGLAQECLH HAQDYGGLLL LATASGNANM VNKLAEGAER 720DGKNNVAFMS YFLQGKVDAC LELLIRTGRL PEAAFLARTY LPSQVSRVVK LWRENLSKVN 780QKAAESLADP TEYENLFPGL KEAFVVEEWV KETHADLWPA KQYPLVTPNE ERNVMEEGKD 840FQPSRSTAQQ ELDGKPASPT PVIVASHTAN KEEKSLLELE VDLDNLELED IDTTDINLDE 900DILDD 905

TABLE 8Human protein: Q9Y678 - Coatomer subunit gamma (Gamma-coat protein)Length: 874 aa, molecular weight: 97718 Da, CRC64Source: http://harvester.embl.de/harvester/Q9Y6/Q9Y678.htmMLKKFDKKDE ESGGGSNPFQ HLEKSAVLQE ARVFNETPIN PRKCAHILTK ILYLINQGEH  60LGTTEATEAF FAMTKLFQSN DPTLRRMCYL TIKEMSCIAE DVIIVTSSLT KDMTGKEDNY 120RGPAVRALCQ ITDSTMLQAI ERYMKQAIVD KVPSVSSSAL VSSLHLLKCS FDVVKRWVNE 180AQEAASSDNI MVQYHALGLL YHVRKNDRLA VNKMISKVTR HGLKSPFAYC MMIRVASKQL 240EEEDGSRDSP LFDFIESCLR NKHEMVVYEA ASAIVNLPGC SAKELAPAVS VLQLFCSSPK 300AALRYAAVRT LNKVAMKHPS AVTACNLDLE NLVTDSNRSI ATLAITTLLK TGSESSIDRL 360MKQISSFMSE ISDEFKVVVV QAISALCQKY PRKHAVLMNF LFTMLREEGG FEYKRAIVDC 420IISIIEENSE SKETGLSHLC EFIEDCEFTV LATRILHLLG QEGPKTTNPS KYIRFIYNRV 480VLEHEEVRAG AVSALAKFGA QNEEMLPSIL VLLKRCVMDD DNEVRDRATF YLNVLEQKQK 540ALNAGYILNG LTVSIPGLER ALQQYTLEPS EKPFDLKSVP LATAPMAEQR TESTPITAVK 600QPEKVAATRQ EIFQEQLAAV PEFRGLGPLF KSSPEPVALT ESETEYVIRC TKHTFTNHMV 660FQFDCTNTLN DQTLENVTVQ MEPTEAYEVL CYVPARSLPY NQPGTCYTLV ALPKEDPTAV 720ACTFSCMMKF TVKDCDPTTG ETDDEGYEDE YVLEDLEVTV ADHIQKVMKL NFEAAWDEVG 780DEFEKEETFT LSTIKTLEEA VGNIVKFLGM HPCERSDKVP DNKNTHTLLL AGVFRGGHDI 840LVRSRLLLLD TVTMQVTARS LEELPVDIIL ASVG 874Human protein: Q9UBF2 - Coatomer subunit gamma-2Length: 871 aa, molecular weight: 97622 DaSource: http://harvester.embl.de/harvester/Q9UB/Q9UBF2.htmMIKKFDKKDE ESGSGSNPFQ HLEKSAVLQE ARIFNETPIN PRRCLHILTK ILYLLNQGEH  60FGTTEATEAF FAMTRLFQSN DQTLRRMCYL TIKEMATISE DVIIVTSSLT KDMTGKEDVY 120RGPAIRALCR ITDGTMLQAI ERYMKQAIVD KVSSVSSSAL VSSLHMMKIS YDVVKRWINE 180AQEAASSDNI MVQYHALGVL YHLRKNDRLA VSKMLNKFTK SGLKSQFAYC MLIRIASRLL 240KETEDGHESP LFDFIESCLR NKHEMVIYEA ASAIIHLPNC TARELAPAVS VLQLFCSSPK 300PALRYAAVRT LNKVAMKHPS AVTACNLDLE NLITDSNRSI ATLAITTLLK TGSESSVDRL 360MKQISSFVSE ISDEFKVVVV QAISALCQKY PRKHSVMMTF LSNMLRDDGG FEYKRAIVDC 420IISIVEENPE SKEAGLAHLC EFIEDCEHTV LATKILHLLG KEGPRTPVPS KYIRFIFNRV 480VLENEAVRAA AVSALAKFGA QNESLLPSIL VLLQRCMMDT DDEVRDRATF YLNVLQQRQM 540ALNATYIFNG LTVSVPGMEK ALHQYTLEPS EKPFDMKSIP LAMAPVFEQK AEITLVATKP 600EKLAPSRQDI FQEQLAAIPE FLNIGPLFKS SEPVQLTEAE TEYFVRCIKH MFTNHIVFQF 660DCTNTLNDQL LEKVTVQMEP SDSYEVLSCI PAPSLPYNQP GICYTLVRLP DDDPTAVAGS 720FSCTMKFTVR DCDPNTGVPD EDGYDDEYVL EDLEVTVSDH IQKVLKPNFA AAWEEVGDTF 780EKEETFALSS TKTLEEAVNN IITFLGMQPC ERSDKVPENK NSHSLYLAGI FRGGYDLLVR 840SRLALADGVT MQVTVRSKER TPVDVILASV G 871

TABLE 9Human protein: P48444 - Coatomer subunit delta (Delta-coat protein)Length: 511 aa, molecular weight: 57210 Da, CRC64Source: http://harvester.embl.de/harvester/P484/P48444.htmMVLLAAAVCT KAGKAIVSRQ FVEMTRTRIE GLLAAFPKLM NTGKQHTFVE TESVRYVYQP  60MEKLYMVLIT TKNSNILEDL ETLRLFSRVI PEYCRALEEN EISEHCFDLI FAFDEIVALG 120YRENVNLAQI RTFTEMDSHE EKVFRAVRET QEREAKAEMR RKAKELQQAR RDAERQGKKA 180PGFGGFGSSA VSGGSTAAMI TETIIETDKP KVAPAPARPS GPSKALKLGA KGKEVDNFVD 240KLKSEGETIM SSSMGKRTSE ATKMHAPPIN MESVHMKIEE KITLTCGRDG GLQNMELHGM 300IMLRISDDKY GRIRLHVENE DKKGVQLQTH PNVDKKLFTA ESLIGLKNPE KSFPVNSDVG 360VLKWRLQTTE ESFIPLTINC WPSESGNGCD VNIEYELQED NLELNDVVIT IPLPSGVGAP 420VIGEIDGEYR HDSRRNTLEW CLPVIDAKNK SGSLEFSIAG QPNDFFPVQV SFVSKKNYCN 480IQVTKVTQVD GNSPVRFSTE TTFLVDKYEI L 511

TABLE 11Human protein: Q9P299 - Coatomer subunit zeta-2 (Zeta-2 coat protein)Length: 210 aa, molecular weight: 23548 Da, CRC64Source: http://harvester.embl.de/harvester/Q9P2/Q9P299.htmMQRPEAWPRP HPGEGAAAAQ AGGPAPPARA GEPSGLRLQE PSLYTIKAVF ILDNDGRRLL  60AKYYDDTFPS MKEQMVFEKN VFNKTSRTES EIAFFGGMTI VYKNSIDLFL YVVGSSYENE 120LMLMSVLTCL FESLNHMLRK NVEKRWLLEN MDGAFLVLDE IVDGGVILES DPQQVIQKVN 180FRADDGGLTE QSVAQVLQSA KEQIKWSLLK 210Human protein: P61923 - Coatomer subunit zeta-1 (Zeta-1 coat protein)Length: 177 aa, molecular weight: 20198 Da,Source: http://harvester.embl.de/harvester/P619/P61923.htmMEALILEPSL YTVKAILILD NDGDRLFAKY YDDTYPSVKE QKAFEKNIFN KTHRTDSEIA  60LLEGLTVVYK SSIDLYFYVI GSSYENELML MAVLNCLFDS LSQMLRKNVE KRALLENMEG 120LFLAVDEIVD GGVILESDPQ QVVHRVALRG EDVPLTEQTV SQVLQSAKEQ IKWSLLR 177

TABLE 10Human protein: O14579 - Coatomer subunit epsilon (Epsilon-coat protein)Length: 307 aa, molecular weight: 34351 DaSource: http://harvester.embl.de/harvester/O145/O14579.htmAPPAPGPASG GSGEVDELFD VKNAFYIGSY QQCINEAQRV KLSSPERDVE RDVFLYRAYL  60AQRKFGVVLD EIKPSSAPEL QAVRMFADYL AHESRRDSIV AELDREMSRS VDVTNTTFLL 120MAASIYLHDQ NPDAALRALH QGDSLECTAM TVQILLKLDR LDLARKELKR MQDLDEDATL 180TQLATAWVSL ATGGEKLQDA YYIFQEMADK CSPTLLLLNG QAACHMAQGR WEAAEGLLQE 240ALDKDSGYPE TLVNLIVLSQ HLGKPPEVTN RYLSQLKDAH RSHPFIKEYQ AKENDFDRLV 300LQYAPSA 307

1.-20. (canceled)
 21. A composition comprising: 1) a complex consistingof a human clathrin triskelion and 2) a therapeutic and/or diagnosticagent, wherein the human clathrin triskelion consists of (i) 3 isolatedclathrin heavy chain (CHC) proteins and (ii) 3 isolated clathrin lightchain (CLC) proteins, wherein the human clathrin triskelion is linked tothe therapeutic and/or diagnostic agent.
 22. The composition of claim21, wherein the therapeutic or diagnostic agent is an imaging contrastagent, imaging tracer agent, radiodiagnostic agent, nucleic acid,oligonucleotide, peptide, carbohydrate, phospholipid, antineoplasticagent, metal, chemical, vaccine, vitamin, hormone, antibiotic, orantibody.
 23. The composition of claim 22, wherein the nucleic acid isan mRNA, RNAi, siRNA, or RNA-induced silencing complex (RISC).
 24. Thecomposition of claim 22, wherein the hormone comprises insulin, thyroidstimulating hormone, thyroid hormone, calcitonin, glucagon, prolactin,or Luteinizing Hormone.
 25. The composition of claim 22, wherein theantibody is a monoclonal antibody.
 26. The composition of claim 21,wherein the therapeutic and/or diagnostic agent is an agent for treatingParkinson's, multiple sclerosis, epilepsy, meningitis, cancer, orAlzheimer's disease.
 27. The composition of claim 21, wherein thetherapeutic and/or diagnostic agent modifies one or more cellularprocesses selected from the group consisting of: endocytosis,exocytosis, mitosis, cellular trafficking, and cellular signaling. 28.The composition of claim 21, wherein the composition crosses the bloodbrain barrier in less than 30 minutes.
 29. The composition of claim 21,wherein each CHC protein comprises amino acid residues 1-1675 of SEQ IDNO:
 1. 30. The composition of claim 21, wherein the CLC proteins are LCaor LCb.
 31. The composition of claim 21, which does not comprise anadaptor protein.
 32. The composition of claim 31, wherein the adaptorprotein is selected from the group consisting of: AP1, AP2, AP3, AP180,epsin protein, and b-arrestin.
 33. The composition of claim 21, whereinthe CHC is linked to the therapeutic and/or diagnostic agent viapolyethylene glycol (PEG), biotin-avidin interaction, cross-linking, orcovalent bonding.
 34. The composition of claim 22, wherein the imagingcontrast agent comprises a gadolinium-based contrast agent, magneticiron oxide, or manganese-based contrast agent.
 35. The composition ofclaim 34, wherein the gadolinium-based contrast agent comprises achelating agent selected from the group consisting of: Diethylenetriamine pentaacetic acid (DTPA),1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid trisodium salt(DO3A), and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA).
 36. The composition of claim 21, wherein the therapeutic and/ordiagnostic agent comprises manganese, iron, quantum dots, photonic dots,nitroxide labels, transition metal ions, magnetic nanoparticles,paramagnetic lanthanide, optical elements in charge-coupled devices(CCDs) or complementary metal oxide semiconductor (CMOS), iron oxideparticles, luminescent genes, light sensitive agents, photosensitiveagents, or radiodiagnostic agents.
 37. The composition of claim 21,wherein the CHC is linked to one or more targeting moieties.
 38. Thecomposition of claim 37, wherein the one or more targeting moietiescomprises antibodies, peptides, fluorophores, permeation enhancers,amino sugars, lipoproteins, toxins, transferrin, and glycoproteins. 39.The composition of claim 21, wherein the CHC is linked to one or morebiodegradable controlled-release polymers.
 40. A compositioncomprising: 1) a complex consisting of a human clathrin triskelion and2) a therapeutic and/or diagnostic agent, wherein the human clathrintriskelion consists of 3 isolated clathrin heavy chain (CHC), whereinthe human clathrin triskelion is linked to the therapeutic and/ordiagnostic agent
 41. A method of delivering a therapeutic and/ordiagnostic agent into the CNS of a subject, the method comprisingadministering to the subject a composition comprising: 1) a complexconsisting of a human clathrin triskelion and 2) the therapeutic and/ordiagnostic agent, wherein the human clathrin triskelion consists of (i)3 isolated clathrin heavy chain (CHC) proteins and (ii) 3 isolatedclathrin light chain (CLC) proteins, wherein the human clathrintriskelion is linked to the therapeutic and/or diagnostic agent.